CN104204811A - Soluble MANF in pancreatic beta-cell disorders - Google Patents

Soluble MANF in pancreatic beta-cell disorders Download PDF

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CN104204811A
CN104204811A CN201380016366.1A CN201380016366A CN104204811A CN 104204811 A CN104204811 A CN 104204811A CN 201380016366 A CN201380016366 A CN 201380016366A CN 104204811 A CN104204811 A CN 104204811A
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beta cell
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anf
cell
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CN104204811B (en
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浦野文彦
金藏孝介
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University of Massachusetts UMass
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Abstract

The invention provides, in part, methods for diagnosing a pancreatic beta-cell disorder, predicting a subject's risk of developing a pancreatic beta-cell disorder, monitoring pancreatic beta-cell function or pancreatic beta-cell mass in a subject at risk of developing a pancreatic beta-cell disorder, monitoring efficacy of a treatment of a pancreatic beta-cell disorder in a subject, identifying a subject having an increased risk of developing a pancreatic beta-cell disorder, selecting a subject for treatment of a pancreatic beta-cell disorder, selecting a subject for participation in a clinical study, and detecting endoplasmic reticulum stress in a pancreatic beta-cell. These methods include determining at least one level of soluble mesencephalic astrocyte-derived neurotrophic factor (MANF) in a biological sample from the subject. Also provided are pharmaceutical compositions containing a soluble MANF protein and kits containing an antibody or an antigen-binding antibod fragment that binds specifically to a soluble MANF.

Description

Soluble M ANF in the disorder of pancreas beta cell
PRIORITY CLAIM
The application requires the right of priority of the U.S. Patent Application Serial Number 61/590,021 of submission on January 24th, 2012.In this integral body by U.S. Patent Application Serial Number 61/590,021, introduce with for referencial use.
Background technology
Experimenter's the function of pancreas beta cell or the disappearance of quantity are facilitated the morbidity (pathogenesis) of the various diseases that comprises type 1 diabetes (diabetes (diabetes mellitus)), diabetes B and Wolfram syndrome etc.In type 1 diabetes, patient is because insulin deficit has hyperglycaemia level.In general, the absolute shortage of insulin occurs in the patient who suffers from type 1 diabetes, and the relative shortage of insulin occurs in the patient who suffers from diabetes B.Increasing evidence show functional pancreas beta cell group (β-cell mass) reduce be 1 type and diabetes B the two, and diabetes are as the common trait of the syndromic mode of inheritance of the Wolfram (people such as Pipeleers, Novartis Found Symp.292:19-24,2008).Between the progressive stage of 1 type or diabetes B, pancreas Instreptozotocin Induced and group (mass) decline gradually, finally cause insulin deficit and hyperglycemia.Newest research results shows that " stress " pancreas beta cell is to dysfunction and dead responsive (people such as Oslowski, Curr.Opin.Endocrinol.Diabetes Obes.17:107-112,2010; The people such as Oslowski, Curr.Opin.Cell Biol.23:207-215,2011; The people such as Fonseca, Trends Endocrinol.Metab.22:266-274,2011).Auxiliary prediction experimenter will contribute to treatment to the diagnostic marker of the disorderly and dead neurological susceptibility of development pancreas Instreptozotocin Induced or delay the progress of pancreas beta cell disorderly (for example, 1 type or diabetes B) in experimenter.
Summary of the invention
The present invention is based in part on following discovery: stress pancreas beta cell produce and secrete the neurotrophic factor (MANF) in solubility midbrain astroglia source; soluble M ANF protection pancreas beta cell avoids the apoptosis of er stress-induction, and soluble M ANF keeps the endoplasmic reticulum redox homeostasis in pancreas beta cell.
In view of these discoveries, for example, in diagnosis experimenter, pancreas beta cell is disorderly, prediction experimenter is developed the disorder of pancreas beta cell risk, monitoring experimenter (, the experimenter in the disorderly risk of development pancreas beta cell) pancreas Instreptozotocin Induced or pancreas beta cell group is provided herein, differentiates that experimenter that the risk of development pancreas beta cell disorder increases is, for example select the experimenter for the treatment of pancreas beta cell disorder,, select to participate in the experimenter of clinical research and the method (in-vitro method) of the er stress in detection pancreas beta cell.These methods comprise at least one level (for example, the endogenous levels of soluble M ANF in the biological sample from experimenter or in nutrient culture media) of determining soluble M ANF.
(for example also provide the method for the pancreas beta cell disorder of diagnosing experimenter herein, in-vitro method), described method comprises the level of neurotrophic factor (MANF) in the biological sample from experimenter of determining solubility midbrain astroglia source; Level by soluble M ANF in biological sample is compared with the reference levels of soluble M ANF; With by compare experimenter that the level of soluble M ANF in biological sample raise with reference levels, be accredited as that to suffer from pancreas beta cell disorderly.
Also provide herein prediction experimenter develop the disorder of pancreas beta cell risk method (for example, in-vitro method), described method comprises: the level of the neurotrophic factor (mesencephalic astrocyte-derived neurotrophic factor, MANF) of determining solubility midbrain astroglia source in the biological sample from experimenter; Level by soluble M ANF in biological sample is compared with the reference levels of soluble M ANF; With the risk of comparing experimenter that the level of soluble M ANF in biological sample raise with reference levels and be accredited as the disorder of development pancreas beta cell is increased, or the level of soluble M ANF in biological sample reduces or not have the experimenter of significant difference to be accredited as the risk of development pancreas beta cell disorder normal or reduce by comparing with reference levels.
(for example also provide the method for pancreas Instreptozotocin Induced or pancreas beta cell group in monitoring experimenter herein, in-vitro method), described method comprises: determine neurotrophic factor (MANF) in when point very first time solubility midbrain astroglia source level in the biological sample from experimenter; Determine the level of soluble M ANF in the biological sample from experimenter when the second time point; When during by the second time point, the level of soluble M ANF in biological sample put with the very first time, the level of soluble M ANF in biological sample compared; Be accredited as with the level of soluble M ANF in biological sample raises when comparing the second time point with the level of when point very first time soluble M ANF in biological sample experimenter that pancreas Instreptozotocin Induced declines or pancreas beta cell reduces, or while comparing the second time point with the level of when point very first time soluble M ANF in biological sample, the level of soluble M ANF in biological sample reduces or is accredited as pancreas Instreptozotocin Induced in experimenter without the experimenter of marked change and do not change or improve, or pancreas beta cell group does not change or increases.
The method (for example, in-vitro method) of the treatment effect of pancreas beta cell disorder in monitoring experimenter is also provided herein.These methods comprise the neurotrophic factor (MANF) determined in when point very first time solubility midbrain astroglia source level in the biological sample from experimenter, determine the level of soluble M ANF in the biological sample from experimenter when the second time point, when by the second time point the level of soluble M ANF in biological sample and the very first time during point level of soluble M ANF in biological sample compare, wherein (i) very first time point is for before treatment, with the second time point be the random time point after initial in treatment, or (ii) very first time point after treatment is initial (following the initiation for the treatment of), with the second time point be later than treatment during or time point afterwards, the level of soluble M ANF in biological sample reduces and shows that this treatment is effective in experimenter when comparing the second time point with the level of when point very first time soluble M ANF in biological sample.
Treatment is also provided herein or delay the method for the disorderly morbidity of pancreas beta cell in experimenter (onset), the method that reduces the er stress in pancreas beta cell (for example, in-vitro method) or reduce or delay the method (for example, in-vitro method) of the apoptotic cell death of er stress-induction in plural pancreas beta cell colony.These methods comprise soluble M ANF from effective dose to experimenter or the apomorphine (apomorpine) of using, or pancreas beta cell or pancreas beta cell colony are contacted with soluble M ANF or apomorphine.In certain embodiments, soluble M ANF comprise with mammal soluble M ANF protein sequence (for example, one of SEQ ID NOS:2 and 4-7 are any) at least 80% (for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) same sequence.In certain embodiments, for example wherein said method comprises administration of apomorphine, and experimenter does not suffer from erectile dysfunction or Parkinson's disease.
When being also provided, use the pharmaceuticals of the disorderly morbidity of in manufacture is used for the treatment of or delays experimenter pancreas beta cell any method of soluble M ANF described herein (the soluble M ANF that for example, comprises the sequence same with SEQ ID NO:2 at least 80%) or apomorphine herein.The separated soluble M ANF (for example, comprising the separated soluble M ANF containing the sequence same with SEQ ID NO:2 at least 80%) or the apomorphine that in treatment or in delaying experimenter, in the disorderly morbidity of pancreas beta cell, use are also provided herein.
Also provide screening to be used for the treatment of herein or delay the disorderly morbidity of pancreas beta cell in experimenter candidate compound method (for example, in-vitro method), the method for the er stress in minimizing pancreas beta cell (for example, in-vitro method) and reduce or delay the method (for example, in-vitro method) of the apoptotic cell death (apoptotic cell death) of er stress-induction in pancreas beta cell.These methods comprise provides pancreas beta cell, pancreas beta cell is contacted with candidate compound, determine the level of the soluble M ANF being produced by pancreas beta cell under the existence of candidate compound, the level of the soluble M ANF being produced by pancreas beta cell is compared with the reference levels of soluble M ANF, and the level of comparing the soluble M ANF being produced by pancreas beta cell with selection to reference levels raises relevant compound as the candidate compound that is used for the treatment of or delays the disorderly morbidity of pancreas beta cell in experimenter.In these methods, the level of comparing the soluble M ANF being produced by pancreas beta cell with reference levels raises and to show that this candidate compound can be to treatment or delay the disorderly morbidity of pancreas beta cell in experimenter, reduce the er stress in pancreas beta cell or reduce or to delay the apoptotic cell death of er stress-induction in pancreas beta cell useful.
The method of screening the method (for example, in-vitro method) that is used for the treatment of or delays the candidate compound of the disorderly morbidity of pancreas beta cell in experimenter, the method that reduces er stress in pancreas beta cell or minimizing or delaying the apoptotic cell death of er stress-induction in pancreas beta cell is also provided herein.These methods comprise provides the mammalian cell of expressing the reporter protein that contains BiP burst, Redox-sensitive fluorescin (for example Redox-sensitive green fluorescent protein) and amino acid sequence KDEL; This cell is contacted with test compound; Determine the amount of the reporter protein being oxidized in cell; With the amount of the reporter protein being oxidized in cell is compared with reference levels; The level of wherein comparing the reporter protein being oxidized in cell with reference levels raises and to show that candidate compound can to treatment or to delay experimenter's the disorderly morbidity of pancreas beta cell useful.In certain embodiments, reference levels are at the amount that does not have the reporter protein being oxidized in mammalian cell under candidate agent (candidate agent).In certain embodiments, reference levels are the threshold level of the reporter protein of oxidation.
The method of screening the method (for example, in-vitro method) that is used for the treatment of or delays the candidate compound of the disorderly morbidity of pancreas beta cell in experimenter, the method that reduces er stress in pancreas beta cell or minimizing or delaying the apoptotic cell death of er stress-induction in pancreas beta cell is also provided herein.These methods comprise provides the mammalian cell of expressing the reporter protein that contains BiP burst, Redox-sensitive fluorescin (for example Redox-sensitive green fluorescent protein) and amino acid sequence KDEL; This cell is contacted with test compound; Determine the amount of the reporter protein reducing in cell; With the amount of the reporter protein reducing in cell is compared with reference levels; The level of wherein comparing the reporter protein reducing in cell with reference levels reduces and to show that candidate compound can to treatment or to delay experimenter's the disorderly morbidity of pancreas beta cell useful.In certain embodiments, reference levels are at the amount that does not have the reporter protein reducing in mammalian cell under candidate agent (candidate agent).In certain embodiments, reference levels are the threshold level of the reporter protein of reduction.
Kit is also provided, it (for example comprises specific binding soluble M ANF, human soluble MANF) antibody or antigen-binding antibody (antigen-binding antibody) fragment, and be bonded at least one antibody or the antigen-binding antibody fragment of the protein that selects the group that free insulin, C-albumen and IAPP (IAPP) form.Pharmaceutical composition is also provided, it (for example comprises soluble M ANF albumen, the soluble M ANF albumen that comprises the sequence same with SEQ ID NO:2 at least 80%) and/or apomorphine, with at least one additives (additional agent) that are used for the treatment of the disorder of pancreas beta cell (for example, Pioglitazone (pioglitazone), TUDCA, GLP-1 or DPP-4 inhibitor are (for example, sitagliptin (sitagliptin), vildagliptin (vildagliptin), BMS-477118 (saxagliptin), BI 1356 (linagliptin), dutogliptin (dutogliptin), , gigue row spit of fland (gemigliptin) and Egelieting (alogliptin))).
Term " pancreas beta cell is disorderly " means to comprise that pancreas Instreptozotocin Induced (for example insulin secretion) declines or is present in pancreas beta cell number (the pancreas beta cell group) minimizing of the excreting insulin of the work in experimenter as the disease of its pathogenetic part.In certain embodiments, the disorder of pancreas beta cell is further characterized in that the increase of experimenter's the middle er stress of pancreas beta cell colony (for example plural pancreas beta cell).As described herein, the decline of the decline of pancreas Instreptozotocin Induced, the pancreas beta cell number (pancreas beta cell group) of living or the increase that is present in the er stress in experimenter's pancreas beta cell can be used methods described herein or other method indirect detection known in the art.The limiting examples of pancreas beta cell disorder comprises type 1 diabetes (diabetes), diabetes B and Wolfram syndrome.
Term " pancreas Instreptozotocin Induced " means for example, biologically active (for example, the unique especially activity of pancreas beta cell) for describing the pancreas beta cell of mammal (, people).The limiting examples of pancreas Instreptozotocin Induced comprises the synthetic and secretion of insulin, the synthetic and secretion of synthetic and secretion and the C-peptide of IAPP (IAPP).Synthetic and the secretion that detects insulin, IAPP and C-peptide is known in the art.Pancreas Instreptozotocin Induced also can be used methods described herein and means known in the art (for example, determining blood sugar level and definite glycosated Hb A 1C level) indirect detection.
Term " pancreas beta cell group " means the sum of the pancreas beta cell of excreting insulin for example, in mammal (, people) alive.Recorded herein for indirectly determining the method for experimenter's pancreas beta cell group.For example, for indirectly determining that other method of experimenter's pancreas beta cell group is (, determining blood sugar level and definite glycosated Hb A 1C level) known in the art.
Pancreas beta cell group can represent that the sum of pancreas beta cell of experimenter's endogenous work maybe can represent that the quantity of pancreas beta cell of experimenter's endogenous work adds the summation of quantity of the pancreas beta cell (the pancreas beta cell of for example, autotransplatntation, homograft or heteroplastic work) of the work being implanted in experimenter.
Term " soluble M ANF " means to comprise the protein of the sequence same with the solubility mammal form at least 80% of the neurotrophic factor (MANF) in midbrain astroglia source.For example, soluble M ANF can be comprise with SEQ ID NOS:2 and 4-7 any, for example, with the protein of the sequence of the total length at least 80% of SEQ ID NOS:2 and 4-7 same (, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% is same).In certain embodiments, soluble M ANF can be wild type mammal soluble M ANF (for example, SEQ ID NO:2 and 4-7).
Soluble M ANF albumen can be used as treatment processing (for example,, as using the recombinant of any method described herein or the endogenous protein of purifying) and uses.The soluble M ANF albumen of purifying can be for example dry weight purity at least 80% (for example, at least 85%, 90%, 95% or 99%).Recorded other modified forms of soluble M ANF herein.
The level that term " increase " or " rising " mean for example, to measure when the time point early or with reference levels or same experimenter (, at the biological sample from same experimenter) is compared, and observable, can detect or significant level increases.
Term " reduce (decline) " mean with reference levels or same experimenter (for example, at the biological sample from same experimenter) level measured when the time point early or compares, observable, can detect or significant level reduces (decline).
Term " to the soluble M ANF level relevant compound that raises " means induction or the mammalian cell that causes contact with this compound exists, produces or the level of the soluble M ANF (for example protein or mRNA) of secretion with do not exist under this compound, contrast that mammalian cell (for example, the mammalian cell of same or same type) exists, the level of generation or the soluble M ANF (for example protein or mRNA) that secretes compares the compound of rising.
The phrase risk of disease " development " mean with contrast experimenter or colony (for example, health volunteer or colony or there is no experimenter or the colony of the disorderly family history of pancreas beta cell) compare experimenter future will the disorder of development pancreas beta cell relative possibility.Provide definite experimenter to develop the method for the risk (in future) of pancreas beta cell disorder herein, described method comprises the level of determining soluble M ANF.
Term " treatment " comprise reduce experimenter disease (for example, seriousness, duration or the frequency of one or more symptoms of the quantity of the symptom disorder of pancreas beta cell) or minimizing experimenter's disease (for example, pancreas beta cell is disorderly).Term treatment also can comprise and reduces the generation that experimenter is developed the risk (in future) of pancreas beta cell disorder or delayed one or more symptoms of experimenter's pancreas beta cell disorder.
The time span that phrase " delay pancreas beta cell disorderly morbidity " means to observe in experimenter before one or more symptoms of pancreas beta cell disorder increases.In certain embodiments, experimenter can be accredited as the risk increase of development pancreas beta cell disorder in advance.As described herein, experimenter can use methods described herein or for example, by observing the family history of pancreas beta cell disorderly (, diabetes B), be accredited as the risk increase of development pancreas beta cell disorder.
Term " biological sample " comprises any sample that comprises biofluid (biological fluid) gathering from experimenter.In non-limiting sample, biological sample can comprise blood, serum, blood plasma, urine, cerebrospinal fluid, saliva, bile, gastric juice or breast milk.
Phrase " er stress " mean reactive oxygen species (pro-oxidant class (pro-oxidant species)) generation, and cell or organelle unbalance to the endoplasmic reticulum between the ability of the removing toxic substances (removing) of reactive oxygen species (or their intermediate), this reactive oxygen species causes the accumulation of false folding in the movement of oxidation-reduction potential in endoplasmic and/or endoplasmic or unfolded protein.Er stress trigger unique stress path, be called unfolded protein and reply (UPR) (describing in addition in this article).
Er stress in pancreas beta cell can for example, be caused by many molecular events (, in endoplasmic reticulum, the level of the increase of the level of the excessive generation of the increase of the level of fatty acid, hyperinsulinemia (hyperinsulemia), VEGF, hypoxemia, glucose deprivation (glucose deprivation), IAPP mutant, igf mutant body, IL-1, IFN-γ increases or virus infections).Various different chemical reagent also can be used for inducing er stress (for example, thapsigargin or tunicamycin).Er stress has shown that the environment from well-oxygenated environment to reduction more moves by endoplasmic reticulum.In certain embodiments, have by endoplasmic reticulum under ER stressed condition from the reagent of the ability that moves to well-oxygenated environment of reduction by help to reduce ER stress and/or can reduce or prevent ER stress-apoptotic cell death of induction.
Er stress can be used various distinct methods known in the art to detect.Recorded herein for detection of, reduce or delay the illustrative methods that er stress in pancreas beta cell occurs.
Phrase " pancreas beta cell colony " means two or more pancreas beta cells.In certain embodiments, pancreas beta cell colony can be present in mammal (for example, people) experimenter (for example, experimenter's endogenous pancreas beta cell, the autograft of pancreas beta cell, alloplast or xenograft).In certain embodiments, pancreas beta cell colony can cultivate (tissue cultivation) in vitro.In certain embodiments, pancreas beta cell colony is pancreas beta cell system (for example, those pancreas beta cell systems described herein).In certain embodiments, pancreas beta cell can be derived from any mammalian species (for example, people, monkey (for example chimpanzee), mouse, pig, rat or ape).In certain embodiments, pancreas beta cell colony can be primary cell line or immortalized cell system.
Term " pancreas beta cell " means to be conventionally present in the insulin-generation cell in the pancreas islet in mammalian pancreas.As used herein, term pancreas beta cell comprises that the pancreas beta cell that is present in mammalian body (for example, endogenous pancreas beta cell, or the autograft of pancreas beta cell, alloplast or xenograft) or the pancreas beta cell of in vitro culture is (for example, from (ex vivo) in the external rear body of elder generation of the pancreas beta cell of any mammalian species of recording herein (for example, primary) culture or pancreas beta cell system (for example, primary or immortalized cell system).In certain embodiments, the pancreas beta cell being present in mammal exists in pancreas.In certain embodiments, the pancreas beta cell being present in mammal is arranged in depancreatize tissue (for example,, in liver organization) in addition.In other embodiments, pancreas beta cell is for example encapsulated in, in the device (, biocompatible polymer) of implanting in experimenter.Term pancreas beta cell also comprises for example, for example, pancreas beta cell in mammal (, people, pig, rat and mouse) clone or primary mammal (, people, pig, rat and mouse) cell culture.In certain embodiments, pancreas beta cell can come genetic manipulation for example, to express one or more recombinant proteins (, insulin) and/or to reduce the expression of one or more intrinsic proteins with molecular biotechnology.
Term " apoptotic cell death of endoplasmic reticulum-induction " for example means, by the apoptosis that stress trigger in cell (, pancreas beta cell) endoplasmic reticulum.In certain embodiments, for example, unfolded protein in the er stress inducing cell of apoptosis-induced cell death (, pancreas beta cell) is replied (UPR) path.Recorded the typical composition of UPR path herein.As recorded herein, er stress can for example, be caused by plurality of reagents (, biological and chemical reagent).Recorded herein for detection of, measure, reduce and delay the method that the apoptotic cell death of the endoplasmic reticulum-induction in pancreas beta cell occurs.Other method for detection of the apoptotic cell death with measurement endoplasmic reticulum-induction is known in the art.
Term " is determined level " and (is for example meant to use one or more science and technology, molecular biology, molecular genetics, immunology and biochemical method or test) evaluate the level (for example,, in biological sample or cell culture medium) of specific molecular.Phrase determines that level comprises and one or more reagent of specific molecular (for example, the antigen-binding fragment of antibody or antibody) specific binding and the physical contact of sample (for example, biological sample or cell culture medium).
Term " the second time point " generally means any time point for example, occurring afterwards at very first time point (, admission time (time of admission)).What the second time point can occur in after very first time point occurs for for example at least 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 2 months, 6 months, 1 year or 2 years.In certain embodiments, experimenter can implement treatment between very first time point and the second time point.
For a change its photoluminescent property is (for example for term " fluorescin of isotope of redox-sensitive ", at its redox environment, (for example change, the change of the redox environment of endoplasmic reticulum) time, excite and/or emission spectrum (for example, peak excites or emission wavelength) changes) protein.In certain embodiments, the change of this protein fluorescence character can be for example because formation and/or the fracture of more than one disulfide bond occurs.The fluorescin limiting examples of isotope of redox-sensitive comprises the yellow fluorescence protein (rxYFP) of redox oxidation-sensitive type green fluorescent protein (roGFP) and isotope of redox-sensitive.The fluorescin of other isotope of redox-sensitive is known in the art.
Other definition runs through the disclosure and appears in context.Unless otherwise defined, otherwise all technology of using herein and scientific terminology and those skilled in the art the implication of understanding is identical conventionally.Method and material have been recorded herein for using in the present invention; Also can use other applicable method and material known in the art.Material, method and example are only for illustrative is not intended to limit.All communiques of mentioning herein, patented claim, patent, sequence, data base entries (database entries) and other document are with its whole introducing with for referencial use.At conflict in the situation that, this instructions, in comprising and being defined in, will regulate and control.
Further feature of the present invention and advantage, from following detailed description and diagram, and will be apparent from claim.
Accompanying drawing explanation
Fig. 1 need not or process after 24 hours MANF albumen in the enrichment medium from pancreas in rat beta cell system (INS-1 832/13) culture or from pancreas in rat beta cell, is being the Western blotting of the level in (INS-1 832/13) lysate with thapsigargin (50nM) or tunicamycin (0.5 μ g/mL) for being illustrated in.
Fig. 2 is the figure that the relative expression in the pancreas in rat beta cell system (INS-1832/13) that MANF mRNA cultivates is under the following conditions shown: 11mM glucose (24 hours); 25mM glucose (24 hours); 25mM glucose (48 hours); 25mM glucose (72 hours); 11mM glucose and 5 μ M tunicamycins (TM) (24 hours); Or 11mM glucose and 20nM thapsigargin (Tg) (24 hours).The data that illustrate are used quantitative PCR in real time to produce (n=3, S.D.).
Fig. 3 is cultivating with 5mM glucose (LG), 11mM glucose (MG) or 25mM glucose (HG) five days for MANF, WFS1, TXNIP and CHOP mRNA are shown, or cultivates five days and with the figure of relative expression in 0.5 μ M thapsigargin (TG) the cultivation mouse primary island (primary islets) of 6 hours with 5mM glucose (LG).The data that illustrate are used quantitative PCR in real time to produce (n=3, S.D.).
Fig. 4 for illustrate MANF mRNA with 5.5mM glucose (UT), 25mM glucose (HG), 0.25 μ M thapsigargin (TG), 10 μ M IAPPs (IAPP) or 500 μ M containing the palmitate (ester) of bovine serum albumin(BSA) (Palmitate) within 24 hours, process after the figure of relative expression in the people's primary island from two mankind's donors (HP11139-01 and HI 11-20) (human primary islet).The data that illustrate are used quantitative PCR in real time to produce (n=3, S.D.).
Fig. 5 is the Western blotting that the level after soluble M ANF processes without other processing or with 25mM glucose (24 hours), 0.25 μ M thapsigargin (24 hours) or 0.5mM palmitate (ester) (24 hours) in the nutrient culture media of people's primary island is shown.
Fig. 6 is the Western blotting of level that the soluble M ANF of the immunoprecipitation after processing without other processing or with 25mM glucose (24 hours), 0.25 μ M thapsigargin (24 hours) or 0.5mM palmitate (ester) (24 hours) is shown from the nutrient culture media of people's primary island.In this experiment, people MANF is used the anti-human MANF antibody of rabbit (Proteintech) immunoprecipitation, and identical antibody is used for carrying out Western blotting.
Fig. 7 has in the Muridae pancreas beta cell system (MIN6) of one of three kinds of different siRNA molecules (siMANF#1, siMANF#2 or siMANF#3) of negative control siRNA (NC) or target MANFmRNA in the processing without other or with 2 relative expressions of μ M both tunicamycin treatment after 24 hours figure in transfection for BiP mRNA is shown.These data produce (n=3, S.D.) by quantitative PCR in real time.
Fig. 8 be Caspase (caspase)-3 that cracking is shown untreated or with 1 μ M tunicamycin or 100nM thapsigargin process 24 hours and further with 0,40 or the Muridae pancreas beta cell processed of 200nM soluble M ANF be the Western blotting of the level in (MIN6).
Fig. 9 A is the figure of GFP (MEROS-GFP) of the isotope of redox-sensitive of mammal endoplasmic reticulum-location.
Fig. 9 B is two confocal images of COS7 cell.A left side illustrates the fluorescence of MEROS-GFP, the fluorescence of DsRed-ER tracer agent shown in right figure (tracker).
The excitation spectrum (emission wavelength be 510nM) of Figure 10 A after to be one group of MEROS-GFP process in non-processor or with the DTT of variable concentrations.
The emission spectrum (excitation wavelength be 394nM) of the MEROS-GFP that Figure 10 B is oxidation in untreated NSC34 cell.
The MEROS-GFP that Figure 10 C is reduction emission spectrum (excitation wavelength is 473nM) in the NSC34 cell of processing with 2mM DTT.
Figure 11 is one group four the confocal microscopy images that use the MEROS-GFP that 476-nm excitation wavelengths (two width figure below) or 405-nm excitation wavelength (two width figure above) assemble in the INS-1832/13 cells of processing with (right side two width figure) or (left side two width figure) useless 2mM DTT.
Figure 12 for do not add processing (unmarked) or use H 2o 2(1 or 0.1mM H 2o 2) or INS-1 832/13 cell processed of DTT (0.1,0.2,0.5,1,2,5 or 10mM DTT) in the figure of MEROS-GFP ratio.MEROS-GFP is than using microplate reader (plate reader) to determine.The data that illustrate are mean value ± S.D. (n=3).
Figure 13 is not for adding (the left figure) of processing or with 4-the acetylaminohydroxyphenylarsonic acid 4 '-talan maleimide (maleimidylstilbene)-2 in the lysate of the cell of (the right figure) of 2mM DTT processing, irreducibility (non-reducing) the polyacrylamide gel electrophoresis photo of the MEROS-GFP of 2 '-disulfonic acid (AMS)-modification.Lysate for not adding (β ME) (top) of processing or (below) of processing with beta-mercaptoethanol (+β ME) before electrophoresis.
Figure 14 illustrates the figure that processes the time-histories (time course) of MEROS-GFP ratio in the NSC34 cell then washing for 10 minutes with 1mM DTT.MEROS-GFP is than within last minute, calculating to each minute interval of washing between latter four minutes in processing.The line of below represents the fluorescence of observation under the exciting of 473nM, and black line represents MEROS-GFP ratio, and light grey line represents the fluorescence (n=3 of 394nM under exciting; S.D.).
Figure 15 is fluoroscopic assist cell sorting (FACS) data that stable transfection has INS-1 832/13 cell after processing in non-processor or with DTT (0.2,0.5 or 10mM DTT) of MEROS-GFP.Data are used 488nM and the excitation wavelength of 405nM and the emission spectrum of 510nM to collect.From below to top, different data sets is untreated, 0.2mM DTT, 0.5mM DTT and 10mM DTT (showing that the state to reduction more moves after the DTT with increasing concentration processes).
Figure 16 is the histogram that stable transfection has the FACS data of INS-1 832/13 cell after processing in non-processor or with DTT (0.2mM, 0.5mM or 10mM DTT) of MEROS-GFP.Transverse axis represents MEROS-GFP ratio, and the longitudinal axis represents cell number.The cell that peak from left to right represents is untreated, 0.2mM DTT-processes, 0.5mM DTT-processes processes with 10mM DTT.
Figure 17 is the figure that stable transfection has the intermediate value MEROS-GFP ratio in INS-1 832/13 cell after processing in non-processor or with DTT (0.2,0.5, or 10mM DTT) of MEROS-GFP.The MEROS-GFP value illustrating is standardized as untreated value.
Figure 18 be illustrate stable transfection have MEROS-GFP in non-processor or use 1mM H 2o 2process the two width figure of data of the facs analysis of INS-1 832/13 cell after 30 minutes.Right figure is untreated or uses 1mM H 2o 2the histogram of MEROS-GFP ratio in INS-1 832/13 cell of processing.A left side illustrates untreated (left side) or use 1mM H 2o 2process the intermediate value MEROS-GFP ratio in INS-1 832/13 cell of 30 minutes.
Figure 19 for use facs analysis in non-processor or with tunicamycin (TM) (5 μ M; 24 hours), thapsigargin (TG) (10nM; 24 hours), TG (1 μ M; 4 hours), brefeldin A (BreA) (100ng/mL; 24 hours) or MG132 (100nM; 24 hours) figure of the intermediate value MEROS-GFP ratio that calculates in INS-1 832/13 cell after processing.Error line (error bars) expression ± S.D. (* p<0.01).Data standard turns to the intermediate value MEROS-GFP ratio of the untreated cell of calculating.
Figure 20 illustrates the histogram that does not add processing or process the MEROS-GFP ratio in the INS-1832/13 cell of 4 hours with 1 μ M thapsigargin (TG).
Figure 21 is for being used the figure of the intermediate value MEROS-GFP ratio calculating in processing INS-1 832/13 cell after 24,48 or 72 hours with 11mM glucose (24 hours) processing or 25mM glucose of facs analysis.Represent ± S.D of error line.Data standard turns to the intermediate value MEROS-GFP ratio with the 11mM glucose processing cell of 24 hours of calculating.
Figure 22 is for being used the figure of the intermediate value MEROS-GFP ratio calculating in INS-1832/13 cell non-processor, after serum consumption or glucose consumption of facs analysis.Represent ± S.D. of error line (* p<0.01).Data standard turns to the intermediate value MEROS-GFP ratio of the untreated cell of calculating.
Figure 23 for do not add processing or with palmitic acid (0.2mM; 24 hours), palmitic acid (0.2mM) and high glucose (25mM) (24 hours), palmitic acid (0.5mM; 24 hours), oleic acid (0.5mM; 24 hours) or linoleic acid (0.5mM; 24 hours) figure of intermediate value MEROS-GFP ratio in INS-1 832/13 cell processed.Represent ± S.D. of error line data standard turns to the intermediate value MEROS-GFP ratio of the untreated cell of calculating.
Figure 24 for do not add processing or employment IAPP (IAPP) (10 μ M; 24 hours), mouse IAPP (10 μ M; 24 hours) or cell factor (interleukin-1 ' beta ' (5ng/mL), IFN γ (100ng/mL) and TNF α (25ng/mL); 24 hours) figure of intermediate value MEROS-GFP ratio in INS-1 832/13 cell processed.Represent ± S.D. of error line data standard turns to the intermediate value MEROS-GFP ratio of the untreated cell of calculating.
Figure 25 is for from not adding (left side) for processing or (PA) processing the two width figure of FACS data of INS-1 832/13 cell of 24 hours with 0.5mM palmitate (ester).Fluorescence after y axle represents to use 488nM to excite, the fluorescence after x axle represents to use 405nM to excite.
Figure 26 is used facs analysis in non-processor or with 0.5mM palmitate (ester) (PA) or the figure of the MEROS-GFP ratio calculating in INS-1 832/13 cell of 0.5mM oleic acid (OA) processing after 24 hours for illustrating.The data of the cell of processing with palmitate (ester) move right.
Figure 27 is that the cell that reduction is shown is processing 24 hours or processing with 25mM glucose the figure of the number percent in INS-1 832/13 cell colony after 24,48 or 72 hours with 11mM glucose.Represent ± standard deviation of error line (* * p<0.01).
Figure 28 be cell that reduction is shown non-processor, serum consume 24 hours or INS-1 832/13 cell colony of glucose consumption after 24 hours in the figure of number percent.Represent ± standard deviation of error line (* * p<0.01).
Figure 29 illustrates the cell of reduction in non-processor, or with 10 μ M Amylins, 10 μ M mouse IAPP, or the figure of the number percent in INS-1 832/13 cell colony of the combined treatment of interleukin-1 ' beta ' (5ng/mL), IFN γ (100ng/mL) and TNF α (25ng/mL) after 24 hours.Represent ± standard deviation of error line (* * p<0.01).
Figure 30 illustrates the cell of reduction in non-processor, with the figure of the number percent in 0.2mM palmitate (ester), 0.2mM palmitate (ester) and 25mM glucose, 0.5mM palmitate (ester), 0.5mM oleic acid, 0.5mM linoleic acid, bovine serum albumin(BSA) and 0.5mM palmitate (ester), 0.5mM palmitate (ester) and 0.5mM oleic acid and 0.5mM palmitate (ester) and INS-1 832/13 cell colony of 0.5mM linoleic acid processing after 24 hours.Represent ± standard deviation of error line (* * p<0.01).
Figure 31 is the figure of the transfection cell that has BiP-P-mCherry and the figure that the structure of flow cytometer laser rays and wave filter is shown.
The figure that Figure 32 expresses for the intermediate value BiP-P-mCherry in INS-1 832/13 cell of oxidation or reduction.Cell for do not add processing or for using 10nM thapsigargin, 5 μ M tunicamycins, 25mM glucose, serum deprivation (serum deprivation), glucose deprivation (glucose deprivation), 0.5mM palmitate (ester), 10 μ M people IAP, or the combined treatment of interleukin-1 ' beta ' (5ng/mL), IFN γ (100ng/mL) and TNF α (25ng/mL) 24 hours.BiP-P-mCherry expresses and uses facs analysis to determine.Represent ± standard deviation of error line.
Figure 33 is for illustrating the two width histograms with INS-1 832/13 cell of 5 μ M tunicamycins (TM, top histogram) or glucose deprivation (below histogram) the processing oxidation of 24 hours and reduction.X axle represents to express the level by the mCherry of people BiP promoters driven.
Figure 34 is that the 0.5mM palmitate (ester) of using by oneself is processed the histogram of FACS data of INS-1 832/13 cell of 24 hours.
Figure 35 is the histogram that the purity of the oxidation of FACS-separation and the INS-1 of reduction 832/13 cell is shown.
Figure 36 is for illustrating the XBP1 (sXBP) (right figure) of BiP (left figure) and montage at two width figure of the expression with in 0.5mM palmitate (ester) processing INS-1 832/13 cell of 24 hours.The expression of BiP and sXBP is used PCR in real time to measure.Represent ± standard deviation of error line.
Figure 37 is the form that Derlin3, BiP, Herp and the PDla5 expression in the endoplasmic reticulum of purifying from INS-1 832/13 cell oxidation or reduction is shown.Expression data is used DNA microarray analysis to collect.
Figure 38 is for illustrating the number percent (left figure) of dead cell of INS-1 832/13 cell of processing with independent DMSO, 10nM thapsigargin (Tg), 10nM Tg and 10 μ M Pioglitazones or 10nM Tg and 500 μ g/mL cow-bezoar ursodesoxycholic acid (tauroursodeoxycholic acid, TUDCA) or intermediate value MEROS-GFP than the two width figure of (right figure).
Figure 39 is the figure that the screening system of using MEROS-GFP system is shown.
Figure 40 is for illustrating from not having (the picture left above) or having the two width histograms of using the FACS data of the INS-1 832/13-MEROS-GFP cell harvesting after 0.5mM palmitate (ester) is processed (top right plot) 10 μ M apomorphines (Apo), with be illustrated in non-processor, or at the figure existing or do not exist under 10 μ M apomorphines with the number percent of the cell reducing in the INS-1 832/13 MEROS-GFP cell after 0.5mM palmitate (ester) processing.Represent ± standard deviation of error line.
Figure 41 is from using (PA) histogram of the FACS data of the INS-1832/13-MEROS-GFP cell harvesting with Mitoprobe dyeing with 10nM or 50nM apomorphine (Apo) or not processing with 10nM or 50nM apomorphine (Apo) of 0.5mM palmitate (ester).
Figure 42 does not process or with the number percent (left figure) of the low Δ ψ cell that passes through facs analysis classification after independent 0.5mM palmitate (ester) or 0.5mM palmitate (ester) and the processing of 10nM apomorphine and two width figure of the number percent of dead cell for being illustrated in.
Figure 43 is the figure of the number percent of the dead cell after not processing or process with 10nM thapsigargin (Tg) or processing with 10nM Tg and 10nM apomorphine.
Figure 44 is two Western blottings that the amount of the caspase 3 (c-caspase 3) of cracking in INS-1 832/13 cell (shWFS1) (left side Western blotting) that the WFS1 of 2 μ M Doxycyclines (the doxycycline)-processing that is present in the primary human pancreas islet (right side Western blotting) of 10nM thapsigargin-processing and processes with 10nM apomorphine or 10 μ M Pioglitazones knocks out and the PARP (c-PARP) of cracking is shown.
Figure 45 for be illustrated in 11mM glucose or 25mM glucose cultivate after (24 hours) (being respectively upper figure and figure below), and with the figure of the fluoroscopic assist cell sorting data of the number percent of INS1 832/13 cell (pancreatic beta cell system) that contains reduction form EroGFP in the endoplasmic reticulum after 0 or 0.5 μ g/mL soluble M ANF processing 48 hours (being respectively left figure and right figure).Cell is used the wavelength of 473nM and the emission spectrum of 510nM to excite.
Embodiment
The present invention is based on, at least in part based on following discovery, soluble M ANF by stress pancreas beta cell secrete, and soluble M ANF delays the pancreas beta cell apoptotic cell death of er stress-induction and reduces the fluctuation be exposed to the redox state of endoplasmic reticulum in the induction reagent of er stress or the pancreas beta cell of condition.In view of these discoveries, be provided for the treatment effect of pancreas beta cell disorder in for example, in Diagnosis of Pancreatic beta cell is disorderly, prediction experimenter is developed the disorder of pancreas beta cell risk, monitoring experimenter (, the experimenter of the risk in the disorder of development pancreas beta cell) pancreas Instreptozotocin Induced and pancreas beta cell group, monitoring experimenter, differentiate the experimenter that the risk of development pancreas beta cell disorder increases, select the experimenter for the treatment of pancreas beta cell disorder, select to participate in the experimenter of clinical research and the method for the er stress in detection pancreas beta cell.These methods comprise at least one level (for example,, in the biological sample from experimenter or in nutrient culture media) of determining soluble M ANF.
Treatment is also provided or delay experimenter the disorderly morbidity of pancreas beta cell, reduce the er stress in pancreas beta cell or reduce or delay the method for the apoptotic cell death of er stress-induction in plural pancreas beta cell colony.These methods comprise soluble M ANF from effective dose to experimenter or the apomorphine of using, or pancreas beta cell or pancreas beta cell colony are contacted with soluble M ANF.In certain embodiments, for example wherein said method comprises administration of apomorphine, and experimenter does not suffer from erectile dysfunction or Parkinson's disease.
Also provide and screen candidate compound, the er stress in minimizing pancreas beta cell that is used for the treatment of or delays experimenter's the disorderly morbidity of pancreas beta cell or the method that reduces or delay the apoptotic cell death of er stress-induction in pancreas beta cell.In certain embodiments, these methods comprise provides pancreas beta cell, pancreas beta cell is contacted with candidate compound, determine the level of the soluble M ANF being produced by pancreas beta cell under the existence of candidate compound, the level of the soluble M ANF being produced by pancreas beta cell is compared with the reference levels of soluble M ANF.In certain embodiments, these methods comprise provide express contain BiP burst, isotope of redox-sensitive fluorescin (for example, the green fluorescent protein of isotope of redox-sensitive or the yellow fluorescence protein of isotope of redox-sensitive) and the mammalian cell (for example, pancreas beta cell) of the reporter protein of amino acid sequence KDEL; This cell is contacted with test compound; Determine the amount of the reporter protein being oxidized in cell; With the amount of the reporter protein being oxidized in cell is compared with reference levels; The level of wherein comparing the reporter protein being oxidized in cell with reference levels raise show candidate compound can to treatment or delay experimenter the disorderly morbidity of pancreas beta cell, reduce er stress and/or the minimizing in pancreas beta cell or delay the apoptotic cell death of er stress-induction in pancreas beta cell useful.In certain embodiments, these methods comprise provide express contain BiP burst, isotope of redox-sensitive fluorescin (for example, the green fluorescent protein of isotope of redox-sensitive or the yellow fluorescence protein of isotope of redox-sensitive) and the mammalian cell (for example, pancreas beta cell) of the reporter protein of amino acid sequence KDEL; This cell is contacted with test compound; Determine the amount of the reporter protein reducing in cell; With the amount of the reporter protein reducing in cell is compared with reference levels; The level of wherein comparing the reporter protein reducing in cell with reference levels reduce show candidate compound can to treatment or delay experimenter the disorderly morbidity of pancreas beta cell, reduce er stress and/or the minimizing in pancreas beta cell or delay the apoptotic cell death of er stress-induction in pancreas beta cell useful.
Pancreas beta cell is disorderly
Pancreas beta cell is disorderly for being characterised in that experimenter's pancreas Instreptozotocin Induced disorder or the class disease that pancreas beta cell group successively decreases.The pancreas Instreptozotocin Induced that can decline in suffering from the experimenter of pancreas beta cell disorder includes, but not limited to insulin generation and secretion, C-peptide produce and secretion and IAPP (IAPP) generation and secretion.The limiting examples of pancreas beta cell disorder comprises type 1 diabetes (diabetes), diabetes B and Wolfram syndrome.The disorder of pancreas beta cell can occur in the mankind at any age, for example, infant, children, grow up and old experimenter in.For example, the disorder of pancreas beta cell can occur in the age and is greater than in 35,40,45,50,55,60,65,70,75,80,85 or 90 experimenter.
Health care professional (for example, doctor, nurse, doctor assistant or lab technician (laboratory technician)) can for example, by evaluating one or more (, two kinds, three kinds, four kinds or five kinds) symptoms of experimenter's pancreas beta cell disorder, diagnose experimenter disorderly for suffering from pancreas beta cell.The non-limiting symptom of experimenter's pancreas beta cell disorder comprise with healthy individual or colony (for example, fasting blood glucose level is greater than 100mg/dL or is greater than 120mg/dL) (for example compare blood sugar level, fasting blood glucose level) significantly increase, with healthy individual or colony (for example, HbA1 C level is greater than 6.5%, be greater than 7.0%, or be greater than 8.0%) compare glycosylated hemoglobin level (HbA1 C level) and significantly increase, thirsty increase, frequent micturition, ravenous hunger, unaccountable losing weight, there is urine ketone, tired, eye-blurred, sore indolence, mild hypertension, with frequent infection.Health care professional also to a certain extent the disorderly family history of the pancreas beta cell based on experimenter diagnose.Health care professional can for example, diagnose the experimenter who suffers from the disorder of pancreas beta cell when experimenter being sent to medical institutions (, clinic or hospital).In some cases, health care professional can diagnose experimenter disorderly for suffering from pancreas beta cell when experimenter is taken in to medical assistance mechanism.Typically, it is disorderly that doctor diagnoses experimenter's pancreas beta cell after one or more symptoms of observing or detecting experimenter.
Health care professional also can differentiate that experimenter is that the risk of development pancreas beta cell disorder increases based on one or more in following factor: body weight (for example increases, constitutional index (body mass index) >25 or >30), blunt, the family history of pancreas beta cell disorder, race, age, diagnosis suffers from Stein-Leventhal syndrome, hypertension, hdl level (for example declines, lower than 35mg/dL), and high levels of triglycerides (for example, surpassing 250mg/dL).
For the experimenter of Diagnosis of Pancreatic beta cell disorder (for example provide herein, the experimenter of one or more symptoms of performance pancreas beta cell disorder or do not show other method of experimenter's (for example, do not make a definite diagnosis and/or asymptomatic experimenter) of the disorderly symptom of pancreas beta cell.Other method of the experimenter of the risk increase of differentiating the disorder of development pancreas beta cell is also provided.The method for the treatment of experimenter's pancreas beta cell disorder is also provided herein.
The neurotrophic factor (MANF) in midbrain astroglia source
The endogenous levels of soluble M ANF albumen, as described herein, can in any method described herein, detect, for example disorderly as Diagnosis of Pancreatic beta cell, prediction experimenter is developed the risk of pancreas beta cell disorder, monitoring experimenter (for example, the experimenter of the risk in the disorder of development pancreas beta cell) pancreas Instreptozotocin Induced and pancreas beta cell group in, differentiate the experimenter of the risk increase of development pancreas beta cell disorder, select the experimenter for the treatment of pancreas beta cell disorder, select to participate in the experimenter of clinical research, with the label that detects the er stress in pancreas beta cell.Soluble M ANF albumen purifying, separated and/or restructuring also can be used for treatment or delays the morbidity of experimenter's pancreas beta cell disorder, reduces the er stress in pancreas beta cell, and reduces or delay the method for the apoptotic cell death of er stress-induction in plural pancreas beta cell colony.
MANF protein translation is precursor protein, and precursor protein is subsequently for example, from cell (, pancreas beta cell) cracking and be released to soluble protein.Total length (precursor) people's MANF albumen and human soluble MANF albumen are shown below.Below underlined be 25 amino acid signal sequences in precursor people MANF albumen.The mRNA of encoding human precursor MANF albumen is also shown below.
People's precursor MANF albumen (SEQ ID NO:1)
MRRMWATQGLAVALALSVLPGSRALRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFCREARGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEKICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAPKAASARTDL
Human soluble MANF albumen (SEQ ID NO:2)
LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFCREARGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEKICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAPKAASARTDL
People MANF mRNA (SEQ ID NO:3)
ggaggcggtg?cggcgcggcg?ggtgcggttc?agtcggtcgg?cggcggcagc?ggaggaggag?gaggaggagg?aggaggagga?ggatgaggag?gatgtgggcc?acgcaggggc?tggcggtggc?gctggctctg?agcgtgctgc?cgggcagccg?ggcgctgcgg?ccgggcgact?gcgaagtttg?tatttcttat?ctgggaagat?tttaccagga?cctcaaagac?agagatgtca?cattctcacc?agccactatt?gaaaacgaac?ttataaagtt?ctgccgggaa?gcaagaggca?aagagaatcg?gttgtgctac?tatatcgggg?ccacagatga?tgcagccacc?aaaatcatca?atgaggtatc?aaagcctctg?gcccaccaca?tccctgtgga?gaagatctgt?gagaagctta?agaagaagga?cagccagata?tgtgagctta?agtatgacaa?gcagatcgac?ctgagcacag?tggacctgaa?gaagctccga?gttaaagagc?tgaagaagat?tctggatgac?tggggggaga?catgcaaagg?ctgtgcagaa?aagtctgact?acatccggaa?gataaatgaa?ctgatgccta?aatatgcccc?caaggcagcc?agtgcacgga?ccgatttgta?gtctgctcaa?tctctgttgc?acctgagggg?gaaaaaacag?ttcaactgct?tactcccaaa?acagcctttt?tgtaatttat?tttttaagtg?ggctcctgac?aatactgtat?cagatgtgaa?gcctggagct?ttcctgatga?tgctggccct?acagtacccc?catgagggga?ttcccttcct?tctgttgctg?gtgtactcta?ggacttcaaa?gtgtgtctgg?gattttttta?ttaaagaaaa?aaaatttcta?gctgtccttg?cagaattata?gtgaatacca?aaatggggtt?ttgccccagg?aggctcctaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaa
Also recorded ox, rat, mouse, pig, fly and zebra fish soluble M ANF protein sequence, and illustrated below.
Ox soluble M ANF (SEQ ID NO:4)
LRQGDCEVCISYLGRFYQDLKDRDVTFSPASIEKELIKFCREARGKENRLCYYIGATEDAATKIINEVSKPLSHHIPVEKICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAPKAASSRTDL
Soluble rat MANF (SEQ ID NO:5)
LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIEEELIKFCREARGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVE?KICEKLKKKDSQICELKYGECD
Mouse soluble MANF (SEQ ID NO:6)
LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIEEELIKFCREARGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEKICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKELKKILDDWGEMCKGCAEKSDYIRKINELMPKYAPKAASARTDL
Pig soluble M ANF (SEQ ID NO:7)
LRPGDCEVCISYLGRFYQDLKDRDVTFSPASIEKELTKFCREARGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEKICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAPKAASSRTD
Drosophila melanogaster (Drosophila melanogaster) soluble M ANF (SEQ ID NO:8)
LKEEDCEVCVKTVRRFADSLDDSTKKDYKQIETAFKKFCKAQKNKEHRFCYYLGGLEESATGILNELSKPLSWSMPAEKICEKLKKKDAQICDLRYEKQIDLNSVDLKKLKVRDLKKILNDWDESCDGCLEKGDFIKRIEELKPKYSR?SEL
Zebra fish soluble M ANF (SEQ ID NO:9)
LKDGECEVCVGFLQRLYQTIQENNVKFDSDSIEKALLKSCKDAKGKENRFCYYIGATSDAATKITNEVSKPMSYHVPVEKICEKLKKKDSQICELKYDKQVDLSSVDLKKLKVKDLKKILEEWGESCKGCVEKSDFIRKINELMPKYAPSAAKARTDL
Mammal soluble M ANF albumen height is conservative, and wherein people and Niu soluble M ANF albumen have 96% homogeneity, and people and mouse soluble MANF albumen have 99% homogeneity, and people and pig soluble M ANF albumen have 97% homogeneity.The homogeneity of human soluble MANF protein liquid and zebra fish soluble M ANF albumen shared 72% and 88% similarity.
Diagnostic method
The method of diagnosis experimenter's pancreas beta cell disorder is provided herein.These methods comprise the level of definite (analysis) soluble M ANF in the biological sample from experimenter, and the level in biological sample is compared with the reference levels of soluble M ANF by soluble M ANF.In these methods, compare the level of soluble M ANF in biological sample with the reference levels of soluble M ANF and raise that to show that experimenter suffers from pancreas beta cell disorderly, compare with the reference levels of soluble M ANF that the level of soluble M ANF in biological sample reduces or it is disorderly without marked change, to show that experimenter does not suffer from pancreas beta cell.
As mentioned in the arbitrary place of this paper, " reference levels of soluble M ANF " can be the threshold level of soluble M ANF, soluble M ANF (is for example present in contrast experimenter or colony, be not diagnosed as experimenter or the colony (health volunteer or colony) of suffering from disease, one or more symptoms without the disorder of pancreas beta cell, and/or do not there is the family history of pancreas beta cell disorder) in level, or the level of soluble M ANF when same experimenter's early time point.
The level of soluble M ANF can be used methods known in the art to determine.For example, the level of soluble M ANF can be used utilization and the multiple technologies known in the art (for example, enzyme linked immunosorbent assay) of the antibody of soluble M ANF specific binding to detect.With the Multiple Antibodies of human soluble MANF specific binding be (for example, the anti-human soluble M ANF antibody of rabbit, can be purchased from Sigma-Aldrich and the Proteintech) being obtained commercially.
Any method described herein can further comprise from experimenter's acquisition or collect sample (for example, comprising for example biological sample of urine, blood, blood plasma, serum or cerebrospinal fluid of biofluid).In any method described herein, biological sample can before determining the level of soluble M ANF, preserve a period of time (for example, at least 1 hour or at least 24 hours) (for example, 10 ℃ or lower than 10 ℃ at preserve).
Any method described herein can for example, be carried out being sent to the patient of medical institutions (, hospital, clinic or medical assistance mechanism).Experimenter can show one or more symptoms (for example, any symptom of pancreas beta cell described herein disorder) of pancreas beta cell disorder.It is disorderly that experimenter can under a cloudly suffer from pancreas beta cell.Experimenter also can show without the disorderly symptom (asymptomatic experimenter) of pancreas beta cell or only have the disorderly symptom of a kind of pancreas beta cell.Experimenter can have the family history of pancreas beta cell disorderly (for example, diabetes B).The risk that experimenter also can have the disorder of development pancreas beta cell increases.Experimenter can be infant, children and adolescents, adult or the elderly.
In certain embodiments, described method can detect or observable pancreas beta cell group and/or have can detect or the experimenter of the pancreas Instreptozotocin Induced of observable (for example, not having the pancreas beta cell group that completely loses or the experimenter of pancreas Instreptozotocin Induced) carries out having.Recorded the method that detects pancreas Instreptozotocin Induced herein.Pancreas beta cell group can for example, by observing experimenter's pancreas Instreptozotocin Induced or using methods known in the art (method of, recording in No. 20110123443 communiques of U.S. Patent application) direct-detection.
Diagnostic method described herein can for example, be undertaken by any health care professional (, doctor, lab technician, nurse, physician extenders and Nurse assistant).Diagnostic method described herein can (for example be used in combination with one or more other diagnostic testing process known in the art or described herein, observe or evaluate one or more symptoms of experimenter's pancreas beta cell disorder, for example, the level of ketone in blood sugar monitoring, glycosylated hemoglobin analysis, insulin level, IAPP level, C-peptide level or urine).Diagnostic method described herein can for example, to differentiating that experimenter's (, using any method described herein to differentiate is the experimenter of the risk increase of development pancreas beta cell disorder) of the risk increase that is the disorder of development pancreas beta cell carries out.In certain embodiments, diagnostic method described herein can regularly carry out (for example, at least monthly, every two months, every six months or annual) to differentiating as developing the experimenter of the risk increase of pancreas beta cell disorder.Some embodiment further comprises from experimenter's collection of biological sample.
Some embodiment comprises in addition to differentiating as suffering from pancreas beta cell experimenter disorderly or risk in the disorder of development pancreas beta cell and (for example imposes treatment for the disorder of pancreas beta cell, separated, the soluble M ANF albumen of purifying or restructuring, Pioglitazone, GLP-1 or DPP-4 inhibitor are (for example, sitagliptin, vildagliptin, BMS-477118, BI 1356, dutogliptin, gigue row spit of fland and Egelieting), or arbitrary composition described herein, for example treat the soluble M ANF albumen and/or the apomorphine that contain the sequence same with SEQ ID NO:2 at least 90% of effective dose).Some embodiment comprises that carrying out additional testing confirms the diagnosis of pancreas beta cell disorder in experimenter in addition.Some embodiment comprises selects experimenter for example, for periodicity glucose monitoring (, using the autologous glucose of periodicity (self-glucose) monitoring of blood glucose meter) or any monitoring method described herein.Some embodiment comprises in addition selects experimenter for example, for the periodicity medical evaluation that undertaken by doctor or health care professional (at least every six months, at least once a year, once, at least every three months once, within least every two months, once or at least every month, periodicity was once visited).Some embodiment comprises the result that records diagnostic test in experimenter's medical records, or uses methods described herein to carry out the diagnostic test of pancreas beta cell disorder to being diagnosed as the experimenter's who suffers from the disorder of pancreas beta cell an above member of stem family (lineal family).
The method of the risk of prediction development pancreas beta cell disorder
Also provide prediction experimenter to develop the method for the risk of pancreas beta cell disorder.These methods comprise the level of definite (test) soluble M ANF in the biological sample from experimenter and by soluble M ANF the level in biological sample compare with the reference levels of soluble M ANF.Compare the level of soluble M ANF in biological sample with the reference levels of soluble M ANF and raise and show that experimenter develops the risk of pancreas beta cell disorder and increase, and compare the level of soluble M ANF in biological sample with reference levels and reduce or do not have significant difference to show that experimenter develops the Risk Reduction of pancreas beta cell disorder or not remarkable.Any reference levels of soluble M ANF described herein can be used in these methods.Some embodiment comprises from experimenter and obtains biological sample in addition.
The level of soluble M ANF can be used methods known in the art to determine.For example, the level of soluble M ANF can be used utilization and the multiple technologies known in the art (for example, enzyme linked immunosorbent assay) of the antibody of soluble M ANF specific binding to detect.In certain embodiments, described method comprises in addition from experimenter's acquisition or collects sample (for example, comprising for example biological sample of urine, blood, blood plasma, serum or cerebrospinal fluid of biofluid).
Any method described herein can for example, be carried out being sent to the patient of medical institutions (, hospital, clinic or medical assistance mechanism).In certain embodiments, described method is carried out experimenter as a part for the periodicity physical examination of being undertaken by health care professional.Experimenter can show one or more symptoms (for example, any symptom of pancreas beta cell described herein disorder) of pancreas beta cell disorder.It is disorderly that experimenter can under a cloudly suffer from pancreas beta cell.Experimenter also can show without the disorderly symptom (asymptomatic experimenter) of pancreas beta cell or only have the disorderly symptom of a kind of pancreas beta cell.Experimenter can have the family history of pancreas beta cell disorderly (for example, diabetes B).Experimenter can be infant, children and adolescents, adult or the elderly.
These diagnostic methods described herein can for example, be undertaken by any health care professional (, doctor, lab technician, nurse, physician extenders and Nurse assistant).These methods can with for differentiate the risk in the disorder of development pancreas beta cell experimenter any other method known in the art (for example, evaluate following one or more factor: body weight increases, family history, race, age, diagnosis blunt, the disorder of pancreas beta cell suffer from Stein-Leventhal syndrome, hypertension, hdl level (for example decline, lower than 35mg/dL) and high-caliber triglyceride (for example,, higher than 250mg/dL)) be used in combination.Differentiate that experimenter that the risk for the disorder of development pancreas beta cell increases can use any method monitoring described herein (for example, referring to, next part).
Some embodiment comprises that the experimenter who increases to the risk of differentiating as the disorder of development pancreas beta cell (for example imposes treatment for the disorder of pancreas beta cell in addition, separated, the soluble M ANF albumen of purifying or restructuring, Pioglitazone, GLP-1 or DPP-4 inhibitor are (for example, sitagliptin, vildagliptin, BMS-477118, BI 1356, dutogliptin, gigue row spit of fland and Egelieting), or arbitrary composition described herein, for example treat the soluble M ANF albumen and/or the apomorphine that contain the sequence same with SEQ ID NO:2 at least 90% of effective dose).Some embodiment comprises that the experimenter who selects to differentiate the risk increase that is the disorder of development pancreas beta cell for example, for periodicity glucose monitoring (, using the autologous glucose monitoring of periodicity of blood glucose meter).Some embodiment comprises in addition select to be differentiated as for example having experimenter that the risk of development pancreas beta cell disorder increases, for the periodicity medical evaluation that undertaken by doctor or health care professional (at least every six months, at least once a year, once, at least every three months once, within least every two months, once or at least every month, periodicity was once visited).Some embodiment comprises the test findings recording in experimenter's medical records in addition, or the experimenter's who increases by the risk that method described herein is the disorder of development pancreas beta cell to discriminating an above member of stem family similarly tests or the risk that develops the disorder of pancreas beta cell is determined in any test known in the art.
The method that monitoring pancreas Instreptozotocin Induced is disorderly and pancreas beta cell is rolled into a ball
The method of monitoring experimenter's (for example, the experimenter of the risk in the disorder of development pancreas beta cell, suffers from the experimenter of pancreas beta cell disorder or have the experimenter of the pancreas beta cell graft of reception) pancreas Instreptozotocin Induced disorder is also provided.These methods comprise the level of soluble M ANF in the biological sample from experimenter when definite (test) put in the very first time, determine (test) soluble M ANF level in the biological sample from experimenter when the second time point, and the level of soluble M ANF in biological sample compared when the level of soluble M ANF in biological sample put with the very first time during by the second time point.When while putting with the very first time, the level of soluble M ANF is compared the second time point, the level of soluble M ANF in biological sample raises and shows that experimenter's pancreas Instreptozotocin Induced declines (for example, significant, observable or detectable decline) or the minimizing of pancreas beta cell group.When while putting with the very first time, the level of soluble M ANF in biological sample compared the second time point, the level of soluble M ANF in biological sample reduces or shows that without marked change experimenter's pancreas Instreptozotocin Induced or pancreas beta cell group do not change or increase.
In certain embodiments, described method for example, is carried out the experimenter's (, not having that the pancreas beta cell completely losing is rolled into a ball or the experimenter of pancreas Instreptozotocin Induced at the first and second time points) who has detectable or observable pancreas beta cell group at very first time point and the second time point and/or have a pancreas Instreptozotocin Induced of detectable or observable amount.
In certain embodiments, the second time point can be at least 6 hours (for example, at least 12 hours, 18 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years or 5 years) after very first time point.In certain embodiments, very first time point can be and enters the admission time of medical institutions or occur first in 1 week of at least one symptom of pancreas beta cell disorder.
In certain embodiments, described method can comprise definite (test) soluble M ANF level in the biological sample from experimenter when more than one other time point slightly late in addition.In these methods, with soluble M ANF for example, a little earlier (, at once before) level in sample ((chronological time) collects a little earlier in chronological order) compares, the level of soluble M ANF in slightly slow sample (collecting after a while in chronological order) raises and shows that experimenter's pancreas Instreptozotocin Induced declines (for example, significant, observable or detectable decline) or the minimizing of pancreas beta cell group.Similarly, with soluble M ANF for example, a little earlier (, at once before) level in sample (in chronological order a little earlier collect) compares, the decline of the level of soluble M ANF in sample (collecting after a while in chronological order) slightly late or show that without marked change experimenter's pancreas Instreptozotocin Induced or pancreas beta cell group do not change or increase.In certain embodiments, these methods are to for example, carrying out first, second and the more than one experimenter who has detectable or observable pancreas beta cell group while putting At All Other Times and/or have the pancreas Instreptozotocin Induced of detectable or observable amount (, at first, second and more than onely do not have the pancreas beta cell group that completely loses or the experimenter of pancreas Instreptozotocin Induced while putting At All Other Times).
In certain embodiments, experimenter can accept pancreas beta cell graft in advance, so that these methods are monitored pancreas Instreptozotocin Induced and the pancreas beta cell group of the pancreas beta cell being implanted in experimenter to a certain extent.In certain embodiments, the pancreas beta cell of transplanting is autotransplatntation, homograft or heteroplastic pancreas beta cell.In certain embodiments, the pancreas beta cell of transplanting is present in device, or is surrounded or be placed in biocompatible polymer by biocompatible polymer.In certain embodiments, the pancreas beta cell of transplanting is for example present in, in depancreatize tissue (, hepatic tissue) in addition.In certain embodiments, these methods are transplanted in the experimenter in 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year and are carried out at pancreas beta cell.In certain embodiments, very first time point after graft procedure (for example,, in 1 week or 2 weeks) soon.
The level of soluble M ANF can be used methods known in the art to determine.For example, the level of soluble M ANF can be used utilization and the multiple technologies known in the art (for example, enzyme linked immunosorbent assay) of the antibody of soluble M ANF specific binding to detect.In certain embodiments, described method comprises in addition from experimenter's acquisition or collects sample or at least two samples (for example, comprising for example biological sample of urine, blood, blood plasma, serum or cerebrospinal fluid of biofluid).
Any method described herein can for example, be carried out being sent to the patient of medical institutions (, hospital, clinic or medical assistance mechanism).In certain embodiments, described method is carried out the part as the periodic test being undertaken by health care professional to experimenter.Experimenter can show one or more symptoms (for example, any symptom of pancreas beta cell described herein disorder) of pancreas beta cell disorder.Experimenter also can show without the disorderly symptom (asymptomatic experimenter) of pancreas beta cell or only have the disorderly symptom of a kind of pancreas beta cell.Experimenter can have the family history of pancreas beta cell disorderly (for example, diabetes B).Experimenter can be infant, children and adolescents, adult or the elderly.
These methods described herein can for example, be undertaken by any health care professional (, doctor, lab technician, nurse, physician extenders and Nurse assistant).Differentiate as thering is pancreas Instreptozotocin Induced and (for example decline, significantly, observable or detectable decline) or the experimenter that reduces of pancreas beta cell group for the treatment of pancreas beta cell disorder (for example can impose, separated, the soluble M ANF albumen of purifying or restructuring, Pioglitazone, GLP-1 or DPP-4 inhibitor are (for example, sitagliptin, vildagliptin, BMS-477118, BI 1356, dutogliptin, gigue row spit of fland and Egelieting), or arbitrary composition described herein, for example treat the soluble M ANF albumen and/or the apomorphine that contain the sequence same with SEQ ID NO:2 at least 90% of effective dose).In certain embodiments, for example, wherein said method comprises administration of apomorphine, and experimenter does not have erectile dysfunction or Parkinson's disease.
Some embodiment (for example comprises experimenter's the additional detected of other label of one or more (for example two kinds, three kinds or four kinds) pancreas Instreptozotocin Induced obstacles or evaluation in addition, the IAPP generation of the C-peptide generation reducing and the insulin generation of secreting, reducing and secretion, reduction and secretion, the blood sugar level of increase for example,, the existence of (, in urine) ketone in the glycosylated hemoglobin level and experimenter's biofluid of increase).Method for detection of one or more other labels of pancreas beta cell is known in the art.
Some embodiment comprises in addition to differentiating as having pancreas Instreptozotocin Induced declines or pancreas beta cell group reduces experimenter and (for example imposes treatment for the disorder of pancreas beta cell, separated, the soluble M ANF albumen of purifying or restructuring, Pioglitazone, GLP-1 or DPP-4 inhibitor are (for example, sitagliptin, vildagliptin, BMS-477118, BI 1356, dutogliptin, gigue row spit of fland and Egelieting), or arbitrary composition described herein, for example treat the soluble M ANF albumen and/or the apomorphine that contain the sequence same with SEQ ID NO:2 at least 90% of effective dose).Some embodiment comprises that it is for example to have the experimenter of the decline of pancreas Instreptozotocin Induced or the minimizing of pancreas beta cell group, for periodicity glucose monitoring (, using the autologous glucose monitoring of periodicity of blood glucose meter) that selection is differentiated.Some embodiment comprise in addition select to differentiate for have pancreas Instreptozotocin Induced declines or pancreas beta cell group reduces experimenter for the periodicity medical evaluation by doctor or health professional (at least every six months for example, at least once a year, once, at least every three months once, at least every two months once, at least every month once or at least weekly periodicity visit).Some embodiment comprises the test findings recording in experimenter's medical records in addition, or to differentiating that the experimenter's that pancreas Instreptozotocin Induced declines or pancreas beta cell group reduces an above member of stem family similarly tests or pancreas Instreptozotocin Induced or pancreas beta cell group are monitored in any test known in the art in order to have.Some embodiment comprises that selecting to have the experimenter that pancreas Instreptozotocin Induced declines or pancreas beta cell group reduces transplants for pancreas beta cell in addition.
The method of the treatment effect of monitoring pancreas beta cell disorder
The method of the treatment effect of monitoring experimenter's (for example, being diagnosed as the experimenter who suffers from the disorder of pancreas beta cell) pancreas Instreptozotocin Induced disorder is also provided.These methods comprise the level of soluble M ANF in the biological sample from experimenter when definite (test) put in the very first time, determine (test) soluble M ANF level in the biological sample from experimenter when the second time point, when by the second time point the level of soluble M ANF in biological sample and the very first time during point level of soluble M ANF in biological sample compare, wherein (i) very first time point is for before treatment, with the second time point be the random time point after initial in treatment, or (ii) very first time point after treatment is initial, with the second time point be later than treatment during or time point afterwards, the level of soluble M ANF in biological sample reduces and shows that this treatment is effective in experimenter when comparing the second time point with the level of when point very first time soluble M ANF in biological sample.In certain embodiments, the treatment of pancreas beta cell disorder is for example, in administration of insulin (, the insulin of arbitrary form described herein), Pioglitazone and TUDCA one or more.In certain embodiments, treat as pancreas beta cell being implanted into experimenter's (for example,, as described herein).
When while putting with the very first time, the level of soluble M ANF is compared the second time point, the level reduction of soluble M ANF in biological sample shows that treatment effectively in experimenter.With when point very first time soluble M ANF level while comparing the second time point the level of soluble M ANF in biological sample increase or show to fail to respond to any medical treatment and/or this treatment should stop and/or should use alternative medicine to experimenter without marked change.In certain embodiments, with when point very first time soluble M ANF level while comparing the second time point the level of soluble M ANF in biological sample increase or show to use and to increase therapeutic dose or should be with frequency and/or the duration administering therapeutic improving to experimenter without marked change.
In certain embodiments, described method for example, is carried out the experimenter's (, not having the pancreas beta cell group that completely loses or the experimenter of pancreas Instreptozotocin Induced at the first and second time points) who has detectable or observable pancreas beta cell group when the first and second time points and/or have a pancreas Instreptozotocin Induced of detectable or observable amount.In certain embodiments, described method is carried out having differentiated in advance or be diagnosed as the experimenter who suffers from the disorder of pancreas beta cell.Some embodiment comprises the experimenter who selects to suffer from the disorder of pancreas beta cell in addition.Some embodiment comprises from experimenter and obtains sample in addition.Some embodiment comprises the treatment of using one or more dosage to experimenter in addition.
In certain embodiments, the second time point can be at least 6 hours (for example, at least 12 hours, 18 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years or 5 years) after very first time point.In certain embodiments, very first time point can be and enters the admission time of medical institutions or occur first in 1 week of at least one symptom of pancreas beta cell disorder.
In certain embodiments, described method can further comprise definite (test) soluble M ANF level in the biological sample from experimenter when more than one other time point slightly late.In these methods, with soluble M ANF for example, a little earlier (, at once before) level in sample (in chronological order a little earlier collect) compares, and the level of soluble M ANF in sample (collecting after a while in chronological order) slightly late reduces and shows the treatment effect in experimenter.Similarly, with soluble M ANF for example, a little earlier (, at once before) level in sample (in chronological order a little earlier collect) compares, the rising of the level of soluble M ANF in sample (collecting after a while in chronological order) slightly late or show that without marked change treatment is invalid in experimenter.In certain embodiments, these methods are to for example, carrying out first, second and the more than one experimenter who has detectable or observable pancreas beta cell group while putting At All Other Times and/or have the pancreas Instreptozotocin Induced of detectable or observable amount (, at first, second and more than onely do not have the pancreas beta cell group that completely loses or the experimenter of pancreas Instreptozotocin Induced while putting At All Other Times).
In certain embodiments, experimenter can accept pancreas beta cell graft in advance, so that these methods are monitored pancreas beta cell to a certain extent, is implanted in the effect in experimenter.In certain embodiments, the pancreas beta cell of transplanting is autotransplatntation, homograft or heteroplastic pancreas beta cell.In certain embodiments, the pancreas beta cell of transplanting is present in device, or is surrounded or be placed in biocompatible polymer by biocompatible polymer.In certain embodiments, the pancreas beta cell of transplanting is for example present in, in depancreatize tissue (, hepatic tissue) in addition.In certain embodiments, these methods are transplanted in the experimenter in 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year and are carried out at pancreas beta cell.In certain embodiments, very first time point after graft procedure (for example,, in 1 week or 2 weeks) soon.
The level of soluble M ANF can be used methods known in the art to determine.For example, the level of soluble M ANF can be used utilization and the multiple technologies known in the art (for example, enzyme linked immunosorbent assay) of the antibody of soluble M ANF specific binding to detect.In certain embodiments, described method comprises in addition from experimenter's acquisition or collects sample or at least two samples (for example, comprising for example biological sample of urine, blood, blood plasma, serum or cerebrospinal fluid of biofluid).
Any method described herein can for example, be carried out being sent to the patient of medical institutions (, hospital, clinic or medical assistance mechanism).In certain embodiments, described method is carried out experimenter as a part for the periodicity physical examination of being undertaken by health care professional.Experimenter can diagnose in advance the disorder of pancreas beta cell and/or can show one or more symptoms (for example, any symptom of pancreas beta cell described herein disorder) of pancreas beta cell disorder.Experimenter can be infant, children and adolescents, adult or the elderly.
These methods can for example, be undertaken by any health care professional (, doctor, lab technician, nurse, physician extenders and Nurse assistant).Some embodiment (for example comprises experimenter's the additional detected of other label of one or more (for example two kinds, three kinds or four kinds) pancreas beta cell disorders or evaluation in addition, the C-peptide increasing produces and secretion, the insulin that increase produce and secretion, the IAPP that increase produce and secretion, the blood sugar level reducing, in the glycosylated hemoglobin level and experimenter's biofluid of reduction (for example,, in urine) in the shortage of the second time point or without the ketone of the level of signifiance (respective horizontal with respect to the very first time during point), show that in addition treatment is effective).Method for detection of one or more other labels of pancreas beta cell is known in the art.
The experimenter's that selection is used for the treatment of method
The experimenter's who selects the disorder for the treatment of pancreas beta cell method is also provided.These methods comprise the level of definite (test) soluble M ANF in the biological sample from experimenter; Level by soluble M ANF in biological sample is compared with the reference levels of soluble M ANF; Selection has to be compared experimenter that the level of soluble M ANF in biological sample raise to be used for the treatment of pancreas beta cell disorderly with reference levels.In these methods, do not select to have and compare with reference levels that the level of soluble M ANF in biological sample reduces or the treatment for the disorder of pancreas beta cell without the experimenter of marked change.
Some embodiment can comprise the level of one or more other labels of test or the disorder of definite pancreas beta cell in addition, and wherein the detection of one or more other labels of pancreas beta cell disorder shows to select experimenter to be used for the treatment of the disorder of pancreas beta cell in addition.One or more other labels of these pancreas beta cell disorders comprise that the level of C peptide in the biological sample from experimenter reduces, the level of IAPP in the biological sample from experimenter reduces, the level of insulin in the biological sample from experimenter reduces, one or more blood sugar levels of experimenter increase, experimenter's glycosylated hemoglobin level increases or in the biological sample from experimenter (for example,, in urine) ketone detected.The method of the level for detection of C-peptide, IAPP, insulin, blood sugar, glycosylated hemoglobin and ketone in the biological sample from experimenter is known in the art.
The level of soluble M ANF can be used methods known in the art to determine.For example, the level of soluble M ANF can be used utilization and the multiple technologies known in the art (for example, enzyme linked immunosorbent assay) of the antibody of soluble M ANF specific binding to detect.In certain embodiments, described method comprises in addition from experimenter's acquisition or collects sample (for example, comprising for example biological sample of urine, blood, blood plasma, serum or cerebrospinal fluid of biofluid).Described method can for example, be undertaken by any health care professional (, doctor, nurse, physician extenders, lab technician or Nurse assistant).
Experimenter can show one or more symptoms (for example, any symptom of pancreas beta cell described herein disorder) of pancreas beta cell disorder.Experimenter also can show without the disorderly symptom of pancreas beta cell or only have the disorderly symptom of a kind of pancreas beta cell.Experimenter can under a cloudly suffer from the disorder of pancreas beta cell or experimenter can have the risk increase that develops the disorder of pancreas beta cell.Experimenter can have the family history of pancreas beta cell disorderly (for example, diabetes B).Experimenter can be diagnosed as in advance suffers from the disorder of pancreas beta cell.
In certain embodiments, described method for example, is carried out the experimenter's (, not having the pancreas beta cell group that completely loses or the experimenter of pancreas Instreptozotocin Induced) who has detectable or observable pancreas beta cell group and/or have a pancreas Instreptozotocin Induced of detectable or observable amount.
The treatment of the pancreas beta cell disorder that can use to experimenter includes, but are not limited to: soluble M ANF (the soluble M ANF albumen that for example, contains the sequence same with SEQ ID NO:2 (with SEQ ID NO:2 total length) at least 80%), apomorphine, Semilente Insulin (for example, asparagus fern (aspart) or bad dried meat (lispro) insulin), fugitive (for example, insulin regular), intermediate-acting insulins (for example, middle effect isophane insulin (neutral protamine Hagedorn) or NPH insulin), protamine zine insulin (for example, super slow (ultralente) insulin), insulin glargine (insulin glargine), insulin detemir (insulin detemir), pramlintide (pramlintide), intestines hypoglycemic mimetics (incretin mimetics) (for example, Exenatide (exenatide)), sulfonylurea (for example, chlorpropamide (chorpropamide), Glipizide (glipizide), glibenclamide (glyburide) and Glimepiride (glimepiride)), meglitinides (meglitinides) (for example, Repaglinide (repaglinide) and Nateglinide (nateglinide)), biguanides (for example, melbine), thiazolidinediones (for example, Rosiglitazone (rosiglitazone) and Pioglitazone), alpha-glucosidase restrainer (for example, acarbose (acarbose) and Miglitol (meglitol)) and DPP-4 inhibitor (for example, sitagliptin and BMS-477118).The treatment of pancreas beta cell disorder can comprise one or more (for example, two kinds, three kinds or four kinds) mentioned reagent of using with combination in any.In certain embodiments, for example, wherein said method comprises administration of apomorphine, and experimenter does not suffer from erectile dysfunction or Parkinson's disease.
Some embodiment in these methods comprises in addition to experimenter (for example uses at least one, two kinds, three kinds or four kinds) treatment of the disorderly use of pancreas beta cell (for example, one or more treatments of described herein or pancreas beta cell known in the art disorder).For example some embodiment in these methods comprises at least potion (for example, at least two doses, four doses, six doses, eight doses or ten doses) of using any pharmaceutical composition described herein in addition.Sustainable a period of time for the treatment of of pancreas beta cell disorder, at least 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or 10 years.Treatment can cyclic application to experimenter (for example, once a day, twice of every day, every day three times, every day four times, once in a week, twice weekly, on every Wendesdays time, on every Thursdays time, every month once, twice of every month, every month three times or every month four times).
In certain embodiments, selection have compare with reference levels experimenter that the level of soluble M ANF raises for the periodicity medical evaluation that undertaken by doctor or health professional (at least every six months for example, at least once a year, once, at least every three months once, at least every two months once, at least every month once or at least weekly periodicity visit).Some embodiment comprises the medical records that records experimenter in addition, experimenter should use or prescribe (prescribed) the disorder of pancreas beta cell one or more treatment (for example, any exemplary treatment described herein).In certain embodiments, select to have and compare the experimenter that soluble M ANF level raises with reference levels and transplant for pancreas beta cell.
Differentiate the method for the risk of experimenter in the disorder of development pancreas beta cell
The method of differentiating the risk of experimenter in the disorder of development pancreas beta cell is also provided.These methods comprise determines the level of soluble M ANF in the biological sample from experimenter; Level by soluble M ANF in the biological sample from experimenter is compared with the reference levels of soluble M ANF.If compare the level of soluble M ANF in the biological sample from experimenter with reference levels, raise, experimenter differentiates that the risk for the disorder of development pancreas beta cell increases.If compared with reference levels, the level of soluble M ANF in the biological sample from experimenter reduces or without marked change, experimenter differentiates the Risk Reduction for the disorder of development pancreas beta cell.
In any method described herein, the risk increasing be with respect to do not have soluble M ANF level significant or observable rising experimenter (for example, use any method described herein not to be diagnosed as the experimenter who suffers from the disorder of pancreas beta cell, be not diagnosed as the health volunteer who suffers from disease, or do not there is the experimenter of the symptom of pancreas beta cell disorder or the family history of pancreas beta cell disorder).
The level of soluble M ANF can be used standard method (for example, any method based on antibody known in the art) to determine.Described method can for example, be undertaken by any health care professional (, doctor, nurse, physician extenders, lab technician or Nurse assistant).
Experimenter can show one or more symptoms (for example, any symptom of pancreas beta cell described herein disorder) of pancreas beta cell disorder.It is disorderly that experimenter also can under a cloudly suffer from pancreas beta cell.Experimenter also can show without the disorderly symptom of pancreas beta cell or only have the disorderly symptom of a kind of pancreas beta cell.Experimenter can have the family history of pancreas beta cell disorderly (for example, diabetes B).
In certain embodiments, described method for example, is carried out having experimenter's (, not having the pancreas beta cell group that completely loses or the experimenter of pancreas Instreptozotocin Induced) of the pancreas Instreptozotocin Induced of detectable or observable pancreas beta cell group and/or detectable or observable amount.
Differentiate that the treatment (for example, any treatment described herein) that can impose the disorder of pancreas beta cell for developing the experimenter of the risk increase of pancreas beta cell disorder maybe can impose new or alternative pancreas beta cell disorder treatment.Differentiate as developing the experimenter of the risk increase of pancreas beta cell disorder and also can carry out more positive treatment processing (for example, the cycle of visiting clinic or hospital of increase).
Some embodiment comprises that the experimenter who increases to the risk of differentiating as the disorder of development pancreas beta cell (for example imposes treatment for the disorder of pancreas beta cell in addition, separated, the soluble M ANF albumen of purifying or restructuring, Pioglitazone, GLP-1 or DPP-4 inhibitor are (for example, sitagliptin, vildagliptin, BMS-477118, BI 1356, dutogliptin, gigue row spit of fland and Egelieting), or arbitrary composition described herein, for example treat the soluble M ANF albumen and/or the apomorphine that contain the sequence same with SEQ ID NO:2 at least 90% of effective dose).Some embodiment comprises that the experimenter who selects to differentiate the risk increase that is the disorder of development pancreas beta cell for example, for glucose monitoring (, using the autologous glucose monitoring of blood glucose meter).Some embodiment comprises in addition selects experimenter for example, for the periodicity medical evaluation that undertaken by doctor or health care professional (at least every six months, at least once a year, once, at least every three months once, within least every two months, once or at least every month, periodicity was once visited).Some embodiment comprises the test findings recording in experimenter's medical records in addition, or the experimenter's who increases by the risk that method described herein is the disorder of development pancreas beta cell to discriminating an above member of stem family similarly tests or the risk that develops the disorder of pancreas beta cell is determined in any test known in the art.
Select the experimenter's of participation clinical research method
The experimenter's who selects participation clinical research method is also provided.These methods comprise determines the level of soluble M ANF in the biological sample from experimenter; Level by soluble M ANF in the biological sample from experimenter is compared with the reference levels of soluble M ANF, with selection have for example, compare with reference levels (, any reference levels of soluble M ANF described herein) level of soluble M ANF in biological sample raise or reduce or without the experimenter of marked change for participating in clinical research.
The level of soluble M ANF can be used standard molecular biology method to determine (for example, any method based on antibody described herein).Described method can for example, be undertaken by any health care professional (, doctor, nurse, physician extenders, lab technician or Nurse assistant).
In certain embodiments, experimenter can show one or more symptoms (for example, any symptom of pancreas beta cell described herein disorder) of pancreas beta cell disorder.In certain embodiments, experimenter also can show without the disorderly symptom of pancreas beta cell or only have the disorderly symptom of a kind of pancreas beta cell.In certain embodiments, experimenter can have the family history of pancreas beta cell disorderly (for example, diabetes B).In certain embodiments, experimenter can be diagnosed as and suffer from the disorder of pancreas beta cell.In certain embodiments, experimenter is just taking the treatment of pancreas beta cell disorder.In certain embodiments, experimenter imposes new or alternative pancreas beta cell disorder treatment during clinical research.
Detect the method for the er stress in pancreas beta cell
The method that detects the er stress in pancreas beta cell is also provided, described method comprises the level of determining the soluble M ANF being produced by pancreas beta cell, and the level of soluble M ANF and the reference levels of soluble M ANF (for example, any reference levels described herein) that produce are compared.The level of comparing the soluble M ANF being produced by pancreas beta cell with the reference levels of soluble M ANF raises and shows that the er stress in pancreas beta cell increases.The level of comparing the soluble M ANF being produced by pancreas beta cell with the reference levels of soluble M ANF reduce or without marked change show pancreas beta cell do not experience can detection level er stress.
The level of soluble M ANF can be used standard molecular biology method to determine (for example, any method based on antibody described herein).Described method can for example, be undertaken by any health care professional (, doctor, nurse, physician extenders, lab technician or Nurse assistant) or scientist.
In certain embodiments, pancreas beta cell mammal (for example, people) in, and the level of the soluble M ANF being produced by pancreas beta cell can be from for example, from determining in mammiferous biological sample (sample that, comprises blood, serum or blood plasma).In certain embodiments, pancreas beta cell can be the pancreas beta cell (for example, autotransplatntation, homograft or heteroplastic pancreas beta cell) of endogenous pancreas beta cell or transplanting.In certain embodiments, pancreas beta cell is the autoplastic pancreas β-thin of genetic modification.In certain embodiments, pancreas beta cell for example can be present in, in the tissue that maybe can be present in mammalian pancreas beyond depancreatize (, liver).In certain embodiments, pancreas beta cell can be present in device, biocompatible materials or polymkeric substance.
In certain embodiments, pancreas beta cell is present in external (for example,, in tissue culture).For example, the elementary pancreas beta cell gathering from mammal can be cultivated in first external body again.In certain embodiments, the pancreas beta cell of cultivation is elementary mammal (for example, people, rat, monkey, ox or pig) pancreas beta cell system.In certain embodiments, the mammalian pancreas beta cell of cultivating can genetic manipulation (for example, genetic modification is for expressing one or more protein or genetic modification for reducing one or more protein expressions) or chemical treatment (for example, utilizing one or more growth factors).In certain embodiments, pancreas beta cell can for example, be cultivated under the existence of one or more biocompatible polymers or biosynthetic dressing (, auxiliary pancreas beta cell is implanted into for example, polymkeric substance or material in mammal (, people)).Various biocompatible polymers and biosynthetic dressing are known in the art.
In certain embodiments, in pancreas beta cell in subject, one or more following processes are followed in the detection of er stress: the experimenter who differentiates the level increase of er stress in his or her pancreas beta cell, administering therapeutic agent (for example, the reagent of the er stress in pancreas beta cell will be reduced, for example, any soluble M ANF albumen described herein and/or apomorphine); Monitoring experimenter's pancreas Instreptozotocin Induced (for example, any method of monitoring pancreas Instreptozotocin Induced described herein); For example, with increase experimenter's clinical frequency of visiting or the monitoring of the general level of the health (, increasing the frequency of blood sugar test).In certain embodiments, for example, wherein said method comprises administration of apomorphine, and experimenter does not suffer from erectile dysfunction or Parkinson's disease.
In certain embodiments, in vitro detection pancreas beta cell, the increase of er stress (for example then makes pancreas beta cell and therapeutic agent, the reagent of the er stress in pancreas beta cell will be reduced, for example, any soluble M ANF albumen described herein or apomorphine) contact and/or monitor pancreas Instreptozotocin Induced (for example, the method for any monitoring pancreas Instreptozotocin Induced described herein).In any method described herein, pancreas beta cell can utilize at least one other cell type or at least one other clone in vitro culture (for example, cultivate altogether or the cultivation of feeding (feeder culture)).
Methods for the treatment of
The method for the treatment of or delaying experimenter's the disorderly morbidity of pancreas beta cell is also provided, described method comprise to experimenter, use effective dose soluble M ANF (for example, the soluble M ANF albumen of purifying, separation or reorganization (for example, any soluble M ANF albumen described herein)) and/or apomorphine.In certain embodiments, the symptom that treatment can cause pancreas beta cell disorder in experimenter (for example, any in symptom described herein) quantity reduces, or the order of severity of one or more symptoms of pancreas beta cell disorder (for example, any in symptom described herein), intensity or frequency reduce.In certain embodiments, treatment can cause delay (compare with the experimenter who receives treatment, in the experimenter who does not receive treatment, the time of the actual generation of one or more symptoms increases) of morbidity or one or more symptoms.For example, treatment can cause one or more following results: the loss ratio of the ratio reduction that experimenter's blood sugar level reduces, experimenter's glycosylated hemoglobin level reduces, the loss ratio of experimenter's insulin production reduces, experimenter's pancreas Instreptozotocin Induced is lost and experimenter's pancreas beta cell group reduces.In certain embodiments, for example, wherein said method comprises administration of apomorphine, and experimenter does not suffer from erectile dysfunction or Parkinson's disease.
Soluble M ANF
For example, in certain embodiments, the soluble M ANF using to experimenter comprise with SEQ ID NOS:2 and 4-7 any, same (for example with the total length at least 80% of SEQ ID NOS:2 and 4-7, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% is same) sequence, and optionally there is the biologically active (herein record) of soluble M ANF albumen.In certain embodiments, the soluble M ANF using to experimenter comprises with SEQ ID NO:2 (human soluble MANF), same (for example with the total length at least 95% of SEQ ID NO:2, at least 96%, 97%, 98%, 99% or 100% is same) sequence, and optionally there is the biologically active (herein record) of soluble M ANF.In certain embodiments, the soluble M ANF using to experimenter is the soluble M ANF of SEQ ID NO:2 or endogenous (wild type) form.In any of these embodiments, soluble M ANF can be purified or be separated.In any of these embodiments, soluble M ANF can be recombinant protein.
Definite use mathematical algorithm of the homogeneity number percent between the comparison of sequence and two sequences completes.Homogeneity number percent between two amino acid sequences is used Needleman and Wunsch, J.Mol.Biol., 48:444-453,1970) algorithm is determined, it has been incorporated to the GAP program (can obtain on internet gcg.com) in GCG software package, use Blossum 62 matrixes or PAM250 matrix, room weight (gap weight) is 16, and length weight is 1.Homogeneity number percent between two nucleotide sequences is used the GAP program (also can obtain on internet gcg.com) in GCG software package to determine, use NWSgapdna.CMP matrix, room weight is 40, and length weight is 1.
Generally speaking, homogeneity number percent between amino acid sequence as referred to herein is used BLAST 2.0 programs to determine, this program Ke Dui NCBI (National Center for Biotechnology Information, NCBI) website opens to the public.Sequence is relatively used without the comparison in room and is used default parameter (Blossum 62 matrixes, room exist that point penalty (gap existence cost) is 11, the gap penalty of each residue be that 1, λ ratio is 0.85) to carry out.For the mathematical algorithm of blast program, be recorded in the people such as Altschul, Nucleic Acids Research 25:3389-3402, in 1997.
As determined herein, the mammal form of soluble M ANF has the sequence homogeneity of height.It will be recognized by those skilled in the art, generally speaking, in order to obtain, (for example there is biologic activity, treatment or delay experimenter the disorderly morbidity of pancreas beta cell, reduce the er stress in pancreas beta cell or reduce or delay the ability of the apoptotic cell death of er stress-induction in two or more pancreas beta cell colonies) soluble M ANF variant, residue not conservative between the soluble M ANF of various mammalian species can change or remove, and the residue of those high conservatives should not change or remove simultaneously.As known in the art, those skilled in the art can compare the various sequences of mammal soluble M ANF albumen provided herein, thereby differentiate residue and those not conservative residues of those high conservatives.
The biologic activity of various forms of soluble M ANF can be tested by carrying out various biological activity test described herein or known in the art those.For example, the reagent stimulating pancreas beta cell of the er stress in induction pancreas beta cell be tested, also be used to the pancreas beta cell that the biologically active of soluble M ANF albumen (for example, any soluble M ANF albumen described herein) can be cultivated by processing under soluble M ANF exists or lacks.With respect to do not contact and use the cell of agent treated with soluble M ANF, having bioactive soluble M ANF will reduce the amount of the apoptosis of the er stress observed in the cell contact with soluble M ANF and reagent or er stress-induction.For example, with respect to processing without soluble M ANF and with the cell of agent treated, there is one or more labels (induction that the anti-death gene (athanogene)-1 (bag-1) that for example, minimizing glucose-regulated protein-78 (also referred to as grp78 or BiP) or bcl-2-are relevant is expressed that bioactive soluble M ANF can reduce er stress in the cell of using the agent treated of inducing er stress; Reduce and activate, golgiosome transposition (Golgi translocation), the nuclear translocation of protease cracking or activating transcription factor 6 (ATF6); Reduce protein kinase RNA sample endoplasmic reticulum kinases (PERK) activation, oligomeric or autophosphorylation; Reduce the activation of IRE1; Reduce the phosphorylation of eIF2 α; Reduce the introne processing of XBP1 mRNA; Reduce the activation of JNK signal transduction pathway; Prevent activation and the cracking of former (procaspase) 4 of Caspase; For example, with the variation (the use isotope of redox-sensitive type eroGFP protein measurement of, recording in embodiment) that prevents or reduce endoplasmic reticulum redox environment).
The soluble M ANF using to experimenter also can comprise one or more (for example, 2,3,4,5,6,7,8,9,10,11,12,13,14 or 15) and inserts, increases, deletes or modify.For example, soluble M ANF (for example can be covalently bond to chemical part, protein (for example, albumin), the sugar (glycan that the glycan that for example, N-connects or O-connect, mannose for example) (referring to, such as people such as Sola, J.Pharm.Sci.98:1123-1245,2009) or significantly increase the half life period of soluble M ANF in experimenter or (for example increase soluble M ANF, at duration of storage) the polymkeric substance (for example, polyglycol) of thermal stability).Any other parts that also can comprise the cell permeability of HIV tat albumen or increase soluble M ANF albumen for the soluble M ANF albumen of these methods.
It is known in the art using the several method of molecular biology and cell culture technology Restruction albumen (for example, recombinant soluble MANF).For example; by mRNA sequence (for example; contain with the soluble M ANF of SEQ ID NO:3 at least 80% same (for example, the mRNA of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% same sequence) coding can transfection to allowing in bacterium, yeast or the mammalian cell (using protein expressing plasmid or viral vectors) of the transfected cellular expression of soluble M ANF.Useful methods known in the art are collected transfectional cell or nutrient culture media and by recombinant soluble MANF protein purification.Other nucleic acid (mRNA) of other mammal soluble M of Multi-encoding ANF albumen is known in the art.
In certain embodiments, first with any diagnostic method described herein or prediction experimenter described herein or known in the art, any method of the risk in the disorder of development pancreas beta cell is differentiated or selects experimenter to be used for the treatment of.
Experimenter can use at least the soluble M ANF of potion (for example, at least 2,3,4 or 5 doses) (for example, any soluble M ANF albumen described herein) and/or apomorphine.Soluble M ANF and/or apomorphine can be at least once a day (for example, twice of every day, every day three times and every day four times), at least weekly (for example, weekly twice, on every Wendesdays time, on every Thursdays time) and/or at least every month applied once to experimenter.Experimenter can for example, for example, by long-time (, at least one month, two months, six months, 1 year, 2 years, 3 years, 4 years or 5 years) treatment (, cyclic application soluble M ANF).As recorded in detail herein, being applied to experimenter's soluble M ANF and/or the dosage of apomorphine can be by doctor by considering multiple physiologic factor, include but not limited to, experimenter's sex, experimenter's body weight, experimenter's age and the existence of other medical conditions are determined.Soluble M ANF and/or apomorphine can be oral, intravenous injection, intra-arterial injection, hypodermic injection, intramuscular injection, intracranial injection or through being injected into cerebrospinal fluid, be applied to experimenter.Similarly, reagent can be made into solid (for example,, for oral) or physiologically acceptable liquid-carrier (for example, salt solution) (for example, in intravenous, artery, subcutaneous, muscle or the administration of brain ridge).
In certain embodiments, experimenter (for example uses at least one in addition, two kinds, three kinds, four kinds or five kinds) other pancreas beta cell disorder treatment (for example, any treatment of pancreas beta cell described herein disorder, as any insulin described herein).In certain embodiments, soluble M ANF and/or apomorphine and at least one are (for example, two kinds, three kinds or four kinds) other pancreas beta cell disorder treatment (for example prepares together, in the physiologically acceptable damping fluid of whole body administrable or nutrient culture media, prepare) (as, any pharmaceutical composition described herein).
Reduce the method for the er stress in pancreas beta cell
The method that reduces the er stress in pancreas beta cell is also provided.These methods comprise make pancreas beta cell and effective dose one or more (for example, two or three) soluble M ANF (for example, any soluble M ANF albumen described herein, as the restructuring that comprises the sequence same with SEQ ID NO:2 at least 80%, purifying or separated soluble M ANF albumen), apomorphine and Pioglitazone.In certain embodiments, pancreas beta cell (tissue cultivation) in vitro.In certain embodiments, pancreas beta cell can be in experimenter.Pancreas beta cell for these experiments can be any pancreas beta cell described herein.
In certain embodiments, pancreas beta cell can with soluble M ANF, apomorphine and Pioglitazone one or more (for example, two or three) contact long period of time (for example, at least 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 8 hours, 10 hours, 12 hours or 24 hours).In certain embodiments, pancreas beta cell and several doses are (for example, at least two doses, three doses, four doses or five doses) one or more (for example, two or three) of soluble M ANF, apomorphine and Pioglitazone with for example regular time interval (approximately once a day, weekly or every month once), contact.
The minimizing of er stress can be determined (any label that for example, detects or analyze er stress described herein) for detection of the er stress in cell by any means known in the art.The minimizing of one or more labels that for example, the minimizing of er stress can be by er stress in cell detects.In certain embodiments, the minimizing of er stress can be observed by one or more following events: the minimizing that the induction of grp78 (BiP) or bag-1 express; The minimizing of activation, golgiosome transposition, protease cracking or ATF6 nuclear translocation; The minimizing of PERK activation, oligomerization or autophosphorylation; The minimizing of the activation of IRE1; The minimizing of eIF2 α phosphorylation; The minimizing of the introne processing of XBP1 mRNA; The minimizing of the activation of JNK signal transduction pathway; The activation of Caspase former 4 and the minimizing of cracking; And for example, by (being exposed to minimizing that the redox environment of the endoplasmic reticulum that the agent of ER-stress-induced induces moves, compare with the contrast pancreas beta cell that is exposed to the agent of ER-stress-induced but for example, processes without one or more (, two or three) of soluble M ANF, apomorphine and Pioglitazone).The minimizing that the redox environment of endoplasmic reticulum moves can be used isotope of redox-sensitive type dye or protein, and the reporter protein of for example recording in embodiment is measured.In certain embodiments, the minimizing of er stress can compare respectively with respect to the amount of the er stress of not observing or detecting with one or more pancreas beta cells that contact of soluble M ANF, apomorphine and Pioglitazone (for example, in vitro or in experimenter) in pancreas beta cell.In certain embodiments, the minimizing of er stress in pancreas beta cell is with respect to not contacting with soluble M ANF albumen, apomorphine or Pioglitazone but contrast pancreas beta cell with the reagent (as thapsigargin) of induction er stress contacts.
These methods can for example, be undertaken by health care professional (, arbitrary class health care professional described herein) or scientist.
Reduce or delay the method for the apoptosis of er stress-induction
There is the pancreas beta cell of the er stress apoptosis pathway in can active cell.The method that reduces or delay the apoptosis of er stress-induction in two or more pancreas beta cell colony is also provided herein, described method comprises (for example makes the colony of pancreas beta cell and the soluble M ANF of effective dose, any soluble M ANF albumen described herein), apomorphine and Pioglitazone one or more (for example, two or three) contact.
In certain embodiments, pancreas beta cell is (tissue cultivation) in vitro.In certain embodiments, pancreas beta cell can be in experimenter (for example,, in the biocompatible materials of transplanting or polymkeric substance).Pancreas beta cell for these methods can be any pancreas beta cell described herein.In certain embodiments, pancreas beta cell can with soluble M ANF, apomorphine and Pioglitazone one or more (for example, two or three) contact long period of time (for example, at least 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 8 hours, 10 hours, 12 hours or 24 hours).In certain embodiments, pancreas beta cell and several doses are (for example, at least two doses, three doses, four doses or five doses) one or more (for example, two or three) of soluble M ANF, Pioglitazone and apomorphine example for example contact with the regular time interval.
In pancreas beta cell colony, the generation of apoptosis and opportunity can be used any method described herein or those methods known in the art to determine.One or more that make pancreas beta cell colony and soluble M ANF, apomorphine and Pioglitazone (for example, two or three) for example, the number percent in the colony that stands apoptosis (, the apoptosis of er stress-induction) of adjustable pancreas beta cell that contacts declines or delays the generation of apoptosis in pancreas beta cell colony.In certain embodiments, with soluble M ANF, apomorphine and Pioglitazone one or more (for example, two or three) reduction of the apoptosis of er stress-induction or delay for example, to compare with the pancreas beta cell colony of one or more (, two or three) processing without soluble M ANF, apomorphine and Pioglitazone in the cell of processing.In certain embodiments, with soluble M ANF, apomorphine and Pioglitazone one or more (for example, two or three) reduction of the apoptosis of er stress-induction or delay for example, to process with one or more (, two or three) without soluble M ANF, apomorphine and Pioglitazone but the contrast pancreas beta cell colony that for example, contacts with the reagent (thapsigargin) of inducing er stress compares in the cell of processing.
The method that detects apoptotic cell death is well known in the art, include but not limited to, cell Caspase (for example, Caspase former-3 and Caspase former-4) cracking, Hoescht and 7-amino-D actinomycin D picked-up, the dUTP breach end mark test (dUTP nick end labeling assay) of TdT-mediation, and the dyeing of annexin film.The various kits for detection of apoptotic cell death are obtained commercially.
Pharmaceutical composition
Also provide herein and (for example comprise at least one soluble M ANF, any soluble M ANF albumen described herein, as, the soluble M ANF of purifying, separation or reorganization) and/or apomorphine, and at least one other processing for the disorder of pancreas beta cell (for example, any processing of one or more pancreas beta cell described herein disorders, for example, Pioglitazone, TUDCA and any insulin described herein) pharmaceutical composition.
In certain embodiments, composition preparation has pharmaceutical acceptable carrier.Pharmaceutical composition and preparation can parenterals, oral or such as using by topical by gasoloid or transdermal etc.Pharmaceutical composition can be prepared by any way and can use by various unit dosage forms, depends on the patient's condition or disease and ill degree, each patient's overall medical conditions and the preferred using method of gained etc.Medicine preparation and the ins and outs of using are recorded well in scientific literature and patent documentation, referring to for example remington:The? science and Practice of Pharmacy, the 21st edition, 2005.
Pharmaceutical composition provided herein can anyly be prepared for using for physianthropy or veterinary usual manner.Wetting agent, emulsifying agent and lubricant, for example lauryl sodium sulfate and dolomol, and colorant, releasing agent (release agents), smears, sweet taste, local flavor and aromatic, antiseptic and antioxidant also can be present in composition.
The preparation of composition of the present invention comprises that those are applicable to intradermal administration, inhalation, the administration of oral/nasal chamber, topical and/or parenteral.Preparation can be presented and can prepare by the known any method of drug world by unit dosage forms easily.Can be combined with carrier material produce single formulation active component (for example, soluble M ANF and/or apomorphine, and one or more additional treatment agent of pancreas beta cell disorder) amount will change as intracutaneous or suction according to host to be treated, specific mode of administration.The amount of the active component that produces single formulation of can being combined with carrier material will be generally the amount of the compound of generation result for the treatment of (for example, described herein one or more arbitrarily result for the treatment of).
Pharmaceutical preparation of the present invention can be manufactured the known any method in field according to medicine and prepare.This type of medicine can comprise sweetener, flavouring agent, colorant and antiseptic.Preparation can be accepted mixed with excipients with the nontoxic pharmacy that is applicable to manufacture.Preparation can comprise one or more thinning agents, emulsifying agent, antiseptic, buffering agent, excipient etc., and can be such as liquor, pulvis, emulsion, freeze-dried powder, spray, creme, lotion (lotions), controlled release preparation, tablet, pill, gel, on patch, in the medium form of graft, provide.
Medicine preparation for oral use can be used pharmaceutical acceptable carrier well known in the art with suitable and applicable dosage preparation.Examples of such carriers makes medicine to be formulated as the tablet that is applicable to be absorbed by patient, pill, pulvis, sugar-coat agent (dragees), capsule, liquor, lozenge (lozenges), gel, syrup, slurries, supensoid agent etc. with unit dosage forms.Medicine preparation for oral use can be formulated as solid excipient, optionally grinds gained potpourri, and the potpourri of processing granular after adding suitable additional compound as required, thereby obtains tablet or sugar-coat agent core (dragee cores).Applicable solid excipient is carbohydrates (carbohydrate) or protein filler, comprises, such as the carbohydrate that comprises lactose, sucrose, sweet mellow wine or sorbierite etc.; Starch from corn, wheat, paddy rice, potato or other plant; Cellulose is as methylcellulose, hydroxypropyl methylcellulose or sodium carboxymethyl cellulose; With the glue class that comprises gum arabic and tragcanth etc.; And such as the protein of gelatin and collagen etc.Can add disintegrant or solubilizer (solubilizing agents) as crospolyvinylpyrrolidone, agar, alginic acid or its salt are as sodium alginate.Sucking fit capsule (Push-fit capsules) can comprise the activating agent that mixes with filler or cementing agent as lactose or starch, and lubricant is as talcum or dolomol, and stabilizing agent optionally.In soft capsule, activating agent can be dissolved or suspended in applicable liquid as in fat oil, whiteruss or liquid macrogol in the situation that being with or without stabilizing agent.
Waterborne suspension can be applicable to manufacture the potpourri of the excipient that is for example applicable to water-based intracutaneous injection of waterborne suspension in (for example comprise activating agent, soluble M ANF and/or apomorphine, and one or more additional treatments of pancreas beta cell disorder).This type of excipient comprises that suspending agent is as sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragcanth and gum arabic, and spreading agent or wetting agent as naturally occurring phosphatide (for example, lecithin), the condensation product of alkylene oxide (alkylene oxide) and fatty acid (for example, Myrj 45), the condensation product of oxirane and long-chain fatty alcohol (for example, 17 carbon ethyleneoxy group cetanols (heptadecaethylene oxycetanol)), oxirane be derived from fatty acid and hexitol partial ester condensation product (for example, polyoxyethylene 80 sorbitan monooleate), or oxirane and be derived from fatty acid and the condensation product of the partial ester of hexitan (for example, Tween-81).Waterborne suspension also can comprise one or more antiseptics as ethyl or n-propyl p-hydroxybenzoate, one or more colorants, and one or more flavouring agents, and one or more sweeteners are as sucrose, Aspartame or asccharin.Grammol osmotic concentration (osmolarity) regulates preparation by weight.
In certain embodiments, oil base medicine (oil-based pharmaceutical) is for using.Oil-based suspension can by vegetable oil as peanut oil, olive oil, sesame oil or coconut oil in, or mineral oil as whiteruss in; Or the activating agent that suspends in these potpourri is prepared.Referring to, for example United States Patent (USP) 5,716, and No. 928, it has been recorded and has used essential oil or essential oil component for increasing bioavilability and reduce between the individuality of oral hydrophobic pharmaceutical compounds and intraindividual variability (also referring to United States Patent (USP) 5,858, No. 401).Oily suspensions can comprise thickening agent as beeswax, solid paraffin (hard paraffin) or cetanol.Can add sweetener so that good to eat oral formulations to be provided, for example glycerine, D-sorbite or sucrose.These preparations can carry out preservation as ascorbic acid by adding antioxidant.As the example of injectable oiliness carrier, referring to Minto, J.Pharmacol.Exp.Ther.281:93-102,1997.
Pharmaceutical preparation also can be the form of emulsion oil-in-water.Oil phase can be above-mentioned vegetable oil or mineral oil, or these potpourri.Applicable emulsifying agent comprises that naturally occurring glue class is if gum arabic and tragcanth, naturally occurring phosphatide are if soybean lecithin, the ester that is derived from fatty acid and hexitan or partial ester are if the condensation product of sorbitan monooleate and these partial esters and oxirane is as Tween-81.Emulsion also can comprise sweetener and flavouring agent as the preparation of syrup and elixir (elixirs).This type of preparation also can comprise mitigator (demulcent), antiseptic or colorant.In an alternative embodiment, these injectable emulsion oil-in-waters of the present invention comprise paraffin oil, sorbitan monostearate, ethoxylation sorbitan monooleate and/or ethoxylation sorbitan trioleate.
Medical compounds also can by nose or intraocular approach use, comprise that insufflation (insufflation), pulvis and aerosol preparations are (for the example of steroid class inhalant (inhalant), referring to for example Rohatagi, J.Clin.Pharmacol.35:1187-1193,1995; Tjwa, Ann.Allergy Asthma Immunol.75:107-111,1995).
In certain embodiments, medical compounds can, by local approach cutaneous penetration, be made applicator stick, solution, suspending liquid, emulsion, gel, creme, paste, paste, gel, coating, pulvis and gasoloid.
In certain embodiments, medical compounds also can be used as microballoon and is administered for sustained release profile in vivo test.For example, the medicine that microballoon can be injected subcutaneous depot through transdermal is used; Referring to Rao, J.Biomater Sci.Polym.Ed.7:623-645,1995; As biodegradable and injectable gel preparation, referring to for example Gao, Pharm.Res.12:857-863,1995; Or as microballoon for oral use, referring to, Eyles for example, J.Pharm.Pharmacol.49:669-674,1997.
In certain embodiments, medical compounds can parenteral administration, for example, by intravenous (IV), muscle, in peritonaeum or subcutaneous administration, or is applied in experimenter's body cavity, organ lumen, or is applied in experimenter's cerebrospinal fluid.These preparations can comprise the solution that is dissolved in the activating agent in pharmaceutical acceptable carrier.Adoptable carrier and the solvent accepted is water and Ringer's mixture (Ringer ' s solution), or isotonic sodium chloride.In addition, aseptic fixedly oil can be used as solvent or suspending medium.For this purpose, can use and comprise synthetic single-or the fixedly oil (fixed oil) of any gentleness (bland) of double glyceride etc.In addition, fatty acid as oleic acid can be similarly for injectable formulation.These solution are aseptic, and conventionally not containing less desirable material.These preparations can be by conventional known sterilization technology sterilizing.Preparation can be according to for approaching need to comprising pharmacy and can accepting adjuvant material of physiological condition, and for example pH adjusting and buffering agent, toxicity correctives, as sodium acetate, sodium chloride, potassium chloride, lime chloride and sodium lactate etc.The concentration of the activating agent in these preparations can extensively change, and will be mainly based on fluid volume, viscosity and body weight etc., according to the specific application pattern of selected or needs of patients, selects.For IV, use, preparation can be sterile injectable preparation, as sterile injectable water-based or oleagenous suspension.This suspending liquid can be prepared with those applicable dispersions or wetting agent and suspending agent.Sterile injectable preparation also can be the suspending liquid of the acceptable thinning agent of non-toxicity parenteral or solvent as in the solution of 1,3-BDO.Use and can be (the by bolus) of bolus injection or continuous (for example, substantially importing incessantly specific a period of time in blood vessel).
In certain embodiments, medical compounds and preparation can freeze-drying.The stable lyophilized formulations that comprises one or more additional treatments of soluble M ANF and/or apomorphine and the disorder of pancreas beta cell can for example, by comprising soluble M ANF and/or apomorphine and one or more additional treatments and filling agent (bulking agent) sweet mellow wine, trehalose, raffinose and sucrose, or prepared by the solution freeze-drying of its potpourri.The process of preparing stable lyophilized formulations can comprise that the about 2.5mg/mL protein of freeze-drying, about 15mg/mL sucrose, about 19mg/mL NaCl and pH are greater than 5.5 and be less than the solution of 6.5 sodium citrate buffer solution.Referring to for example US2004/0028670.
Composition and preparation can be by carrying out administration with liposome.By using liposome, particularly to target cell special or the surface of liposome carrier ligand of preferred pin to certain organs in addition, technician can focus on activating agent in vivo to sending in target cell.Referring to for example, United States Patent (USP) 6,063,400 and 6,007, No. 839; Al-Muhammed, J.Microencapsul.13:293-306,1996; Chonn, Curr.Opin.Biotechnol.6:698-708,1995; And Ostro, Am.J.Hosp.Pharm.46:1576-1587,1989.
Preparation of the present invention can be used for preventative and/or therapeutic treatment and uses.In certain embodiments, for therapeutic application, composition is to be enough to cure, alleviate or part stops the amount of the clinical manifestation of disorderly or its complication to be applied to the risk in disorder described herein or suffers from the experimenter of disorder described herein; This can be described as treatment effective dose.For example, in certain embodiments, pharmaceutical composition of the present invention is used with the amount of the order of severity, duration or frequency of one or more symptoms that is enough to reduce symptom quantity or reduces experimenter's pancreas beta cell disorder.
The amount that has been suitable for the pharmaceutical composition of this processing is treatment effective dose.Dosage (dosage schedule) and this purposes is effectively measured, be that dosage regimen (dosing regimen) will depend on various factors, comprise that the order of severity of stage, disease or the patient's condition of disease or the patient's condition is, the overall state of patient health and patient's health and age etc.When calculating patient's dosage regimen, mode of administration is also taken into account.
Dosage regimen is also considered pharmacokinetic parameter well known in the art,, the absorptivity of activating agent, bioavailability, metabolism and clearance rate (clearance) etc. (referring to, Hidalgo-Aragones for example, J.Steroid Biochem.Mol.Biol.58:611-617,1996; Groning, Pharmazie51:337-341,1996; Fotherby, Contraception 54:59-69,1996; Johnson, J.Pharm.Sci.84:1144-1146,1995; Rohatagi, Pharmazie 50:610-613,1995; Brophy, Eur.J.Clin.Pharmacol.24:103-108,1983; remington:The Science and Practice of? pharmacy,the 21st edition, 2005).State-of-the art allows clinician to determine disease or the patient's condition of each independent patient's dosage regimen, activating agent and treatment.The enforcement method of the present invention that is dosage and dosage level for the guide providing as the similar composition of medicine can be used as determining dosage regimen, uses is correct and suitable guide.
Can use according to following the single or multiple of preparation that provide, for example: dosage as required and frequency, and patient's tolerance etc.Preparation should be for effective treatment, prevents or improves the patient's condition, disease or symptom and the activating agent of q.s is provided.
In an alternative embodiment, medicine preparation consumption per day for oral use is between every day every kg body weight approximately 1 to 100 or more mg.With oral contrary, be applied in blood flow, to body cavity or to organ lumen and can use lower dosage.Substantially higher dosage can be used for local or Orally administered or uses by pulvis, spray or inhalant.Be actually used in that to prepare the outer method that can administered formulation of parenteral or parenteral will be known or apparent for those skilled in the art, and be recorded in more detail in all publications described as follows etc., remington:The Science and Practice of Pharmacy, the 21st edition, 2005.
Kit
Also provide comprise specific binding at least one antibody of soluble M ANF albumen (as any soluble M ANF albumen described herein) or the antibody fragment of antigen-binding (for example, Fab, F (ab') 2, Fab', scFv, di-scFv or sdAb) and another label of specific binding pancreas beta cell disorder is extremely (for example, insulin, C-albumen and IAPP) at least one (for example, two kinds, three kinds or four kinds) antibody or the kit of antigen-binding antibody fragment.In certain embodiments, the antibody or the antigen-binding antibody fragment that are included in kit are positioned at (for example, enzyme linked immunosorbent assay) on substrate.In certain embodiments, kit can further comprise the soluble M ANF albumen (for example, any soluble M ANF described herein) of separation, purifying or restructuring.In certain embodiments, one or more antibody or antigen-binding antibody fragment are labeled (for example, radioactive isotope, fluorophore or in conjunction with albumen (as avidin (avidin)).These kits can be used for, for example disorderly for Diagnosis of Pancreatic beta cell, differentiate the risk of experimenter in the disorder of development pancreas beta cell, or monitoring experimenter's pancreas Instreptozotocin Induced or pancreas beta cell group.In certain embodiments, kit can comprise the explanation of carrying out any method described herein in addition.
Reporter protein
Method operation report albumen provided herein, described reporter protein in conjunction with albumen (BiP) burst (for example comprises, mouse or people BiP burst), fluorescin (for example, the green fluorescent protein of isotope of redox-sensitive and the yellow fluorescence protein of isotope of redox-sensitive) and the amino acid sequence KDEL (Lys-Asp-Glu-Leu) of isotope of redox-sensitive.In certain embodiments, BiP burst is positioned at N-end, and the fluorescin of isotope of redox-sensitive is positioned at the C-end of BiP burst, and amino acid sequence KDEL is positioned at the C end of the fluorescin of isotope of redox-sensitive.In certain embodiments, two or more independently protein elements (protein elements) in being present in reporter protein (for example, the fluorescin of BiP burst, isotope of redox-sensitive and KDEL sequence) between there is 1 to 100 amino acid (for example, 1 to 50,1 to 40,1 to 30,1 to 25,1 to 20,1 to 15 and 1 to 10 amino acid).The amino acid sequence of the green fluorescent protein of isotope of redox-sensitive is listed below.
The green fluorescent protein of isotope of redox-sensitive (SEQ ID NO:10)
MSKGEELFTG?VVPILVELDG?DVNGHKFSVS?GEGEGDATYG?KLTLKFIVTT?GKLPVPWPTL?VTTFXLQCFA?RYPDHMKRHD?FFKSAMPEGY?VQERTIFFKD?DGNYKTRAEV?KFEGDTLVNR?IELKGIDFKE?DGNILGHKLE?YNYNSHCVYI?VADKQKNGIK?VNFKIRHNIE?DGSVQLADHY?QQNTPIGDGP?VLLPDNHYLC?YQSALSKDPN?EKRDHMVLLE?FVTAAGITHG?MDELYK
Reporter protein described herein can comprise the sequence for example, with SEQ ID NO:10 at least 80% (, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) same (as long as gained protein keeps the fluorescent characteristic of its isotope of redox-sensitive).For example, the sudden change that is introduced into the green fluorescent protein of isotope of redox-sensitive or the yellow fluorescence protein of isotope of redox-sensitive should not comprise the sudden change in halfcystine.In certain embodiments, yellow fluorescence protein (for example, the rxYFP that the fluorescin of isotope of redox-sensitive is isotope of redox-sensitive; Be recorded in the people such as Ostergaard, EMBO J.20:5853-5862,2001).Other case history of the fluorescin of isotope of redox-sensitive is in people (J.Biol.Chem.279:22284-22293,2004) such as the people such as Merksamer (Cell 135:933-947,2008) and Dooley.
Exemplary people and the mouse BiP burst that can be present in reporter protein illustrate below.Reporter protein for method described herein can comprise any mammal BiP burst (for example, people or mouse BiP burst).
In certain embodiments, the nucleic acid of reporter protein and these reporter proteins of coding provides the means of the minor fluctuations in for example, redox environment in sensitive (, significantly improving) detection complete (intact) pancreas beta cell.
People BiP burst (SEQ ID NO:12)
MKLSLVAAMLLLLSAARA
Mouse BiP burst (SEQ ID NO:13)
MMKFTVAAALLLLGAVRA
In certain embodiments, reporter protein contains following sequence, the reporter protein at least 80% same (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% is same) of this sequence and the sequence (illustrating below) that contains SEQ ID NO:14.In certain embodiments, reporter protein is by the nucleic acid coding for example containing, with the sequence of SEQ ID NO:15 at least 80% same (, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% is same).For example, the sudden change in the reporter protein of SEQ ID NO:14 should not comprise the sudden change in halfcystine.The difference sudden change of SEQ ID NO:14 can be tested the fluorescent characteristic of determining whether mutant keeps their redox to rely on by any method described herein.The fluorescent characteristic that the specific redox of the protein that comprises the sequence same with SEQ ID NO:14 (MEROS-GFP) at least 80% relies on is recorded in an embodiment in detail.
MEROS-GFP albumen (SEQ ID NO:14)
MMKFTVVAAALLLLGAVRAEEEDPPVATMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFISTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHKVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLKTCSALSKDPNEKRDHMVLLERVTAAGITHGMDELYKTSGGPPPTGEEDTSEKDEL
MEROS-GFP?mRNA(SEQ?ID?NO:15)
atgatgaagttcactgtggtggcggcggcgttgctgctgctgggcgcggtgcgggccgaggaggaggatccaccggtcgccaccatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttccactactggaaaactacctgttccatggccaacacttgtcactactttcagttatggtgttcaatgcttttcaagatacccagatcatatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactgccacaaggtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgaagacatgctctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagcgcgtaacagctgctgggattacacatggcatggatgaactatacaaaactagtggaggccctcccccaactggtgaagaggatacatcagaaaaagatgagttgtag
The screening technique of candidate agent
Also be provided for the screening technique of the candidate compound of following one or more aspects: treat or delay the morbidity of experimenter's pancreas beta cell disorder, reduce the er stress in pancreas beta cell, and reduce or delay the apoptotic cell death of the er stress-induction in pancreas beta cell.These methods comprise provides pancreas beta cell, pancreas beta cell is contacted with candidate compound, and determine the level of the soluble M ANF being produced by pancreas beta cell under candidate compound exists, and the level of the soluble M ANF being produced by pancreas beta cell is compared with the reference levels of soluble M ANF.In these methods, the level of comparing the soluble M ANF being produced by pancreas beta cell with reference levels raises and shows that test compound can be used for following one or more aspect: treat or delay the morbidity of experimenter's pancreas beta cell disorder, reduce the er stress in pancreas beta cell, and reduce or delay the apoptotic cell death of er stress-induction in pancreas beta cell.
Pancreas beta cell for these methods can be any pancreas beta cell described herein (for example, pancreas beta cell system (for example, any pancreas beta cell system described herein) or elementary pancreas beta cell).
The level of soluble M ANF can be used standard molecular biology method (for example, described herein any take antibody as basic method) to determine.The method can for example, be undertaken by any health care professional (, doctor, nurse, physician extenders, lab technician or Nurse assistant) or scientist.
In certain embodiments, the level that reference levels are the soluble M ANF that produced by pancreas beta cell in the situation that not there is not candidate compound.In certain embodiments, reference levels be present in do not suffer from that pancreas beta cell is disorderly, the level of the soluble M ANF in the experimenter of the symptom without the disorder of pancreas beta cell or the disorderly family history of pancreas beta cell.In certain embodiments, reference levels are the level of the soluble M ANF of generation in from mammiferous elementary pancreas beta cell or mammalian pancreas beta cell system.In certain embodiments, reference levels are the threshold level of soluble M ANF.
Also provide screening to be used for the treatment of or to delay the morbidity of experimenter's pancreas beta cell disorder, reduce the er stress in pancreas beta cell, and/or reduce or delay the method for the candidate compound of the apoptotic cell death of er stress-induction in pancreas beta cell.These methods comprise the mammalian cell (for example, mammalian cell pancreas beta cell or pancreas beta cell system) that the reporter protein of expressing the fluorescin contain BiP burst, isotope of redox-sensitive and amino acid sequence KDEL is provided; Cell is contacted with test compound; Determine the amount of the reporter protein being oxidized in cell; With the amount of the reporter protein being oxidized in cell is compared with reference levels; The level of comparing the reporter protein being oxidized in cell with reference levels raises and shows that candidate compound can be used for treating or delaying experimenter's the disorderly morbidity of pancreas beta cell, reduce the er stress in pancreas beta cell, and/or reduce or delay the apoptotic cell death of er stress-induction in pancreas beta cell.In certain embodiments, reference levels are the amount of the reporter protein of the oxidation in mammalian cell in the situation that not there is not candidate agent.In certain embodiments, cell and candidate agent and induction ER stress reagent the two contact, and reference levels be present in independent with induction ER stress the cell of agent treated in the level of reporter protein of oxidation.Induction ER stress the limiting examples of reagent be recorded in herein.Induction ER stress other example be known in the art.In certain embodiments, reference levels are the threshold level of the reporter protein of oxidation.Cell used can be people, mouse, rat, pig, monkey or ox cell.Cell can be any pancreas beta cell described herein or known in the art.In certain embodiments, reporter protein is SEQ ID NO:14, or the protein that contains the sequence for example, with SEQ ID NO:14 at least 80% (, at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) same.
Also provide screening to be used for the treatment of or to delay the morbidity of experimenter's pancreas beta cell disorder, reduce the er stress in pancreas beta cell, and/or reduce or delay the method for the candidate compound of the apoptotic cell death of er stress-induction in pancreas beta cell.The method comprises the mammalian cell (for example, mammalian cell pancreas beta cell) that the reporter protein of expressing the fluorescin contain BiP burst, isotope of redox-sensitive and amino acid sequence KDEL is provided; Cell is contacted with test compound; Determine the amount of the reporter protein reducing in cell; With the amount of the reporter protein reducing in cell is compared with reference levels; The level of comparing the reporter protein reducing in cell with reference levels raises and shows that candidate compound can be used for treating or delaying experimenter's the disorderly morbidity of pancreas beta cell, reduce the er stress in pancreas beta cell, and/or reduce or delay the apoptotic cell death of er stress-induction in pancreas beta cell.In certain embodiments, reference levels are the amount of the reporter protein of the reduction in mammalian cell in the situation that not there is not candidate agent.In certain embodiments, cell and candidate agent and induction ER stress reagent the two contact, and reference levels be present in independent with induction ER stress the cell of agent treated in the level of reporter protein of reduction.Induction ER stress the limiting examples of reagent be recorded in herein.Induction ER stress other example be known in the art.In certain embodiments, reference levels are the threshold level of the reporter protein of reduction.Cell used can be people, mouse, rat, pig, monkey or ox cell.Cell can be any pancreas beta cell described herein or known in the art.In certain embodiments, reporter protein is SEQ ID NO:14, or the protein that contains the sequence for example, with SEQ ID NO:14 at least 80% (, at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) same.
In certain embodiments, for example, by detecting one or more photoluminescent properties (, detecting the peculiar spectral signature of reporter protein of reduction or oxidised form) of reporter protein in cell, determine.In certain embodiments, the level of the reporter protein of reduction or oxidised form is used fluorescence plate reader or is used fluoroscopic assist cell sorting art (FACS) to determine.
For example, express the pancreas beta cell (for example, INS-1 832/13) of reporter protein and can inoculate (plated) to 6-orifice plate, contact with candidate compound, then by Trypsin Induced, collect.With after phosphate buffered saline (PBS) washing, cell (for example can be suspended in applicable medium, 11mM glucose-hanks buffer salt solution) in, with LSRII (BD), carry out FACS, thereby determine the level of the reporter protein of the reduction be present in cell or oxidised form.
In certain embodiments, fluorescence plate reader can be used for determining the level of the reporter protein of the reduction be present in cell or oxidised form.For example, pancreas beta cell system (as INS-1 832/13 cell) can be seeded in 96-orifice plate and with candidate agent and process.The level of the reporter protein of reduction or oxidised form can be used fluorescence plate reader to detect.In this embodiment, deducting of background signal should be carried out before the level of reporter protein of determining reduction or oxidised form.
In certain embodiments, the protein that reporter protein comprises SEQ ID NO:14 or contains the sequence same with SEQ ID NO:14 at least 80%.In these embodiments, the reporter protein of reduction form has the excitation wavelength of 473nM and the emission wavelength of 510nM, and the reporter protein of oxidised form has the excitation wavelength of 394nM and the emission wavelength of 510nM.
In some embodiment of these methods, can determine and in cell, reduce the ratio of the level of reporter protein of form and the level of the reporter protein of oxidised form.In these methods, the ratio of calculating can be with reference than comparing.With reference to the ratio than can be from the cell of not processing with candidate agent.Reference is than also can be threshold value ratio.In certain embodiments, cell and candidate agent and induction ER stress reagent contact, determine and in cell, reduce the ratio of the level of reporter protein of form and the level of the reporter protein of oxidised form, and by the ratio in cell with from independent with induction ER stress the reference of cell of agent treated than comparing.In these methods, make to be defined as with the candidate agent with reference to than comparing the ratio decline in cell the candidate agent of the pancreas beta cell disorder that is used for the treatment of experimenter.
Some embodiment of said method is further included in the animal model of pancreas beta cell disorder and (for example tests candidate compound, determine whether using of candidate compound can treat one or more symptoms of pancreas beta cell disorder in animal model (for example, reducing the order of severity, frequency or duration) or delay the generation of one or more symptoms of pancreas beta cell disorder in animal model).The non-limiting animal model of diabetes B comprises Zhu Ke obese rat (Zucker fatty rats, ZFR), ob/ob (fat (obese)) mouse, cp (fat (corpulent)) rat, Zhu Ke diabetes endomorphy type (ZDF) rat, gerbil jird (fertile gerbil jird (Psammomys obesus)), is rhesus macaque (obsess rhesus monkeys), KK mouse and KK-A ymouse (being recorded in the people such as Srinivasan, Indian J.Med.Res.125:451-472,2007).The non-limiting animal model of type 1 diabetes comprises diabetes type (NOD) mouse and Biology Breeding (BB) rat (being recorded in the people such as Rees, Diabetic Med.22:359-370,2005) of non-obesity.The order of severity of one or more symptoms of pancreas beta cell disorder or generation can be used method described herein or methods known in the art determine or observe in these animals.
Some embodiment of said method further comprises that apoptotic cell death that whether test candidate compound can reduce or delay the er stress-induction in pancreas beta cell colony (for example, compare with the pancreas beta cell colony of agent treated with induction er stress in the situation that not there is not candidate agent, reduce or delay the apoptotic cell death with the er stress-induction in the pancreas beta cell colony of the agent treated of candidate agent and induction er stress).Method for detection of apoptotic cell death is known in the art and comprises, but be not limited to, cell Caspase (cellular caspases) (for example, Caspase former-3 and Caspase former-4) the picked-up of cracking, Hoescht and 7-amino-D actinomycin D, the dUTP breach end mark test of TdT-mediation, and the dyeing of annexin film.The various kits for detection of apoptotic cell death are obtained commercially.
Some embodiment of said method comprises (for example, whether candidate compound reduces the induction of grp78 (BiP) or bag-1 expression in the induction of testing candidate compound and whether preventing or delay other label of er stress in pancreas beta cell; Reduce and activate, golgiosome transposition, the nuclear translocation of protease cracking or ATF6; Reduce PERK activation, oligomeric or autophosphorylation; Reduce the activation of IRE1; Reduce the phosphorylation of eIF2 α; Reduce the introne processing of XBP1 mRNA; Reduce the activation of JNK signal transduction pathway; Prevent activation and the cracking of former (procaspase) 4 of Caspase; And/or prevent or reduce the variation (for example, using any reporter protein described herein to measure) of endoplasmic reticulum redox environment).Several groups of primers that the expression of BiP and Bag-1 can be used quantitative PCR in real time utilization to be designed to hybridize with the part of BiP or Bag-1 are determined.The expression of BiP, Bag-1, PERK, IRE1, eIF2 α, XBP1 and ATF6 or processing, activation, phosphorylation or celluar localization also can be used with the antibody of BiP, a Bag-1, PERK, IRE1, eIF2 α, XBP1 or ATF6 specific binding and utilize methods known in the art to determine.In certain embodiments, one or more labels of er stress prevent or delay be by the cell of the agent treated with candidate agent and induction er stress with in the situation that not there is not candidate agent with inducing one or more pancreas beta cells of the agent treated of er stress to compare.
The candidate compound of any type can be used for said method.Candidate compound, for example, can be protein, peptide, nucleic acid (as RNA or DNA), mineral compound, lipid, oligosaccharides or its combination in any.The library that can be used for the candidate compound of said method is obtained commercially.
The candidate compound screening can be used any in multiple means in combinatorial libraries method known in the art to obtain, and comprising: biological library; The parallel solid phase of space addressing or solution phase library; The synthetic library method that needs deconvolution (deconvolution); " pearl one compound (one-bead one-compound) " library method; With the synthetic library method of using affinity chromatography to select.Biological library means only limit to peptide library, and other four kinds of means are applicable to peptide, non-peptide oligomer or micromolecular compound library (Lam, Anticancer Drug Des., 12:145,1997).
Example for the synthesis of the method for molecular library is found in document, such as: the people such as DeWitt, Proc.Natl.Acad.Sci.U.S.A., 90:6909,1993; The people such as Erb, Proc.Natl.Acad.Sci.U.S.A., 91:11422,1994; The people such as Zuckermann, J.Med.Chem., 37:2678,1994; The people such as Cho, Science 261:1303,1993; The people such as Carrell, Angew.Chem.Int.Ed.Engl., 33:2059,1994; The people such as Carell, Angew.Chem.Int.Ed.Engl., 33:2061,1994; With the people such as Gallop, J.Med.Chem., 37:1233,1994.
The library of compound can exist in solution (for example, Houghten, Bio/Techniques, 13:412-421,1992) (Lam, or on pearl, Nature, 354:82-84,1991), on chip (Fodor, Nature 364:555-556,1993), on bacterium (United States Patent (USP) 5,223,409), (United States Patent (USP) 5,571,698 on spore; 5,403,484; With 5,223,409), (Scott and Smith, Science, 249:386-390,1990 on (people such as Cull, Proc.Natl.Acad.Sci.U.S.A., 89:1865-1869,1992) or bacteriophage on plasmid; Devlin, Science, 249:404-406,1990; The people such as Cwirla, Proc.Natl.Acad.Sci.U.S.A., 87:6378-6382,1990; And Felici, J.Mol.Biol., 222:301-310,1991).
Embodiment
In embodiment 1. pancreas beta cells soluble M ANF express ER stress during increase
Test to determine whether that the expression of soluble M ANF is induced in replying the pancreas beta cell of er stress.These experiments are used two kinds of different chemical reagent (thapsigargin and tunicamycin) of rodent pancreas beta cell system (INS-1 832/13 and MIN6), elementary mouse and people's pancreas islet and induction er stress to carry out.INS-1 832/13 cell is cultivated in the RPMI-1640 that comprises 10% hyclone, penicillin, streptomysin, Sodium Pyruvate and 0.1% beta-mercaptoethanol.Primary island obtains from Prodo, and is inoculated in the 6-orifice plate of the laminin V that scribbles in advance the generation of 804G cell, and cultivates in having augmented hyclone, nonessential amino acid, Sodium Pyruvate and antibiotic CMRL nutrient culture media.
In the nutrient culture media of the pancreas in rat beta cell system (INS-1 832/13) of the cultivation after processing with thapsigargin (50nM), detect soluble M ANF albumen (Fig. 1).In processing the same cell system after 24 hours with 5 μ M tunicamycins or 20nM thapsigargin, detect the elevated levels (Fig. 2) of MANF mRNA.
Data from the experiment of using mouse primary island to carry out further show, with 0.5 μ M thapsigargin, process the remarkable increase (Fig. 3) that mouse islets causes MANF mrna expression for 6 hours.Second group of experiment used the people's primary island from two mankind's donors to carry out.These data also show the remarkable increase (Fig. 4) that causes MANF mrna expression with thapsigargin (0.25 μ M) handler's pancreas islet, and the generation of soluble M ANF albumen and be released into extracellular matrix (Fig. 5 and 6).
Test to determine whether that soluble M ANF protects pancreas beta cell to avoid er stress.In first group of experiment, MANF expresses one of three kinds of different siRNA constructs of use target MANF mRNA and knocks out.According to the service manual of manufacturer, use Lipofectamine 2000 (lipofectamine) to carry out transfection.Data from these experiments show, when tunicamycin for cell (2 μ M) is processed 24 hours, with with contrast siRNA transfection and comparing by the control cells of the both tunicamycin treatment of par, knocking out that MANF expresses causes er stress increase (as shown in the increase of BiP mRNA level) (Fig. 7).These data show, soluble M ANF plays the effect that prevents or reduce the er stress in pancreas beta cell.
Carry out other experiment and can reduce or delay the apoptosis with the er stress-induction in the pancreas beta cell of the chemical reagent processing of er stress-induction to determine whether soluble M ANF.Data from these experiments show, mice pancreatic beta cell system (MIN6) has reduced with the processing of soluble M ANF Caspase-3 burst size (Fig. 8) of observing in the cell after processing with tunicamycin (1 μ M, 24 hours) significantly.These data show, soluble M ANF can prevent or delay the apoptotic cell death of the er stress-induction in pancreas beta cell.
Embodiment 2. is for monitoring the system of ER redox state
Developed the new system that detects the redox state of ER in cell.In this system, the burst (KDEL) of mouse BiP and mammal ER delay signal (retrieval signal) is attached to respectively N-and the C-end (Fig. 9 A) of the green fluorescent protein (GFP) of isotope of redox-sensitive.Recombinant protein is called MEROS-GFP (GFP of the redox-sensitivity of mammal endoplasmic reticulum-location (Mammalian Endoplasmic Reticulum-Localized RedOx-Sensitive GFP)).Fluorescent microscope is used for confirming that MEROS-GFP is positioned ER (Fig. 9 B).In the complete oxidation of MEROS-GFP in NSC34 cell and the material of reduction (species), show different excitation spectrums, at 394nM and 473nM place, had maximum value (Figure 10 A).NSC 34 cells are cultivated in the DMEM that contains 10% hyclone, penicillin and streptomysin.
Excitation spectrum from two different excitation wavelength 508nM and 510nM is comparable (Figure 10 B and 10C).Confocal microscope analysis confirms, in the cell of processing with strong reductant dithiothreitol (DTT) (DTT), from the fluorescence excitation of 476nM, significantly increases, and the fluorescence slight reduction (Figure 11) at 405nM place.
The self-excitation 473nM that comes that is standardized as wild type untreated cell is called MEROS-GFP ratio to the ratio of the fluorescence of 394nM.MEROS-GFP is than using fluorescence plate reader to determine.In these experiments, INS-1 832/13 cell is seeded on 96-orifice plate with 50,000 cells/well, and cell is used H under each concentration 2o 2or DTT processes 30 minutes, measure the fluorescence that excitation wavelength 473nM and emission wavelength 510nM (for the MEROS-GFP of reduction) locate or excitation wavelength 394nM and emission wavelength 510nM (for the MEROS-GFP of oxidation) locate.MEROS-GFP is than determining after subtracting background signal.Data show, oxygenant H 2o 2do not change MEROS-GFP ratio-show MEROS-GFP 100% oxidized (Figure 12) almost in vivo.On the contrary, DTT processes has increased MEROS-GFP than (Figure 12) in dosage-dependence mode.
Carried out other experiment to confirm that MEROS-GFP is than having reacted its change of redox state in vivo.In these experiments, the redox state use irreducibility SDS-PAGE of MEROS-GFP and mercaptan-alkylating reagent, 4-acetylaminohydroxyphenylarsonic acid 4 '-talan maleimide-2,2 '-disulfonic acid (AMS) combination is monitored.In these experiments, INS-1832/13 cell that do not process or that process with 2mM DTT dissolves with 1 * SDS-PAGE sample buffer of the with or without 2-beta-mercaptoethanol that contains 25mM AMS, at 95 ℃, boil 10 minutes, use SDS-PAGE electrophoresis, and use anti-GFP antibody immunoblotting.As desired, the irreducibility SDS-PAGE of the lysate of the cell of processing from DTT-shows the only migration band (migrating form) more slowly of a MEROS-GFP, shows that DTT processes to have reduced the MEROS-GFP in body (Figure 13) completely.
MEROS-GFP is than monitoring in the cell of also processing at DTT-in different time points.Figure 14 shows that ER can reduce within a few minutes that DTT processes, and returns to well-oxygenated environment at DTT wash-out (washout) in one minute.These results show, the redox state in the ER that MEROS-GFP can be used in real time, live body is monitored mammalian cell.
Flow cytometry is for the MEROS-GFP in precise monitoring body.These data demonstrations, pancreas beta cell is that INS-1 832/13 use DTT processing causes fluorescence 405nM place being excited from 488nM to increase (Figure 15 and 16) than dose dependent.In order further to analyze this observation data, in the cell of processing at DTT-, detect intermediate value MEROS-GFP ratio.Data show that DTT processing has increased intermediate value MEROS-GFP than (Figure 17) in dosage-dependence mode.Yet, H 2o 2process and not change MEROS-GFP than (Figure 18), show MEROS-GFP in ER in foundation level (basal level) complete oxidation almost.Intermediate value MEROS-GFP than also by ER stress experiment and physiology derivant, comprise tunicamycin, thapsigargin, brefeldin A, MG 132 (Figure 19 and 20), chronic high glucose (chronic high glucose) (Figure 21), serum consumes, glucose deprivation (Figure 22), and palmitate (ester), people's IAPP (hIAPP) and inflammatory factor (Figure 23 and 24) increase.
Embodiment 3.ER stress cell in the heterogeneity of ER redox state
Two kinds of different cell colonys are observed after processing INS-1 832/13 cell with palmitate (ester) (the strong ER derivant of pancreatic beta cell) by flow cytometer: a kind of ER state that keeps oxidation, another kind has the highly ER (Figure 25 and 26) of reduction.In order further to study these foreign cell colonies, MEROS-GFP is classified as to " cell of reduction " than the cell that is greater than 2.These two kinds of different cell colonys are also observed the following processing that utilizes the ER stress-induced agent of other known pancreas beta cell, comprise chronic high glucose (Figure 27), serum consumption, glucose deprivation (Figure 28) and Amylin (Figure 29).In stress-induced agent, palmitate (ester) is processed increase maximum in the cell colony that causes having the ER highly reducing.What is interesting is, show in advance unsaturated fatty acid, oleic acid and linoleic acid to the protection of the cell dysfunction of palmitate (ester)-induction, suppressed significantly the ability (Figure 30) of palmitate (ester) foundation reduction ER.These data show ER stress cell in its redox state, be heterogeneous.
Embodiment 4. has the activation that in the cell of ER of induction, unfolded protein is replied (UPR)
Test to study the relation between UPR and the heterogeneity of redox state.In these experiments, redox state and UPR activated water are on average monitored in same cell.In order to reach this target, built the UPR reporter gene mCherry by the coding red fluorescent protein of people BiP promoters driven.BiP promoter comprises three unfolded protein response elements (UPRE) and has shown the activation levels of effective reflection UPR.This BiP-mCherry reporter plasmid transfection is to expressing in INS-1 832/13 cell of MEROS-GFP, and by facs analysis for the MEROS-GFP that monitors at same cell than and through the UPR of BiP-mCherry activation levels (Figure 31).The activation levels of BiP-mCherry and MEROS-GFP than induction ER stress after monitor.In these experiments, INS-1 832/13 cell of expressing MEROS-GFP is seeded in 6-orifice plate, with each compound treatment official hour, then by Trypsin Induced, collects.With after phosphate buffered saline (PBS) washing, cell is resuspended in 11mM glucose-hanks buffer salt solution.Flow cytometry analysis utilizes LSRII (BD) to carry out.
Data show that the cell colony (MEROS-GFP is than >2.0) with reduction ER compares and have higher BiP-mCherry activation levels with the cell that can keep being oxidized ER, have indicated the activation (Figure 32 and 33) of UPR in ER cell colony highly reducing.
Carry out other experiment to confirm these data.In these experiments, there is the cell that is oxidized ER and highly reduces ER and using palmitate (ester) processing rear by FACS sorting (Figure 34 and 35), and use the expression of the XBP-1 of PCR in real time mensuration UPR label BiP and montage.In these experiments, the cell of reduction and oxidation is by FACS sorting, and total RNA extracts by RNeasy kit (Qiagen).Reverse transcriptase and quantitative PCR are used BioRad iQ5 to use SYBR green colouring material to carry out.
Data show BiP and montage XBP-1 expression with there is level in the cell of ER of oxidation and compare increase (Figure 36) in thering is the cell of the ER of reduction highly.These data further confirm (Figure 37) by the express spectra having in the cell of the ER of reduction and the ER of oxidation.These experiments are used INS-1 832/13 cell that utilizes 0.5mM palmitate (ester) to process the FACS-sorting of 24 hours to carry out.Total RNA is used RNAeasy kit (Qiagen) to extract.Then the RNA of purifying is used for to GeneChip Rat Gene 1.0 ST Array (Affymetrix) according to the service manual of manufacturer.These data show to nourish the highly cell of the ER of reduction and also have overactive UPR, may be as a kind of regulation mechanism.
The little molecular screening of embodiment 5. shifts ER compound from reducing environment to well-oxygenated environment from
Above-mentioned data show, the disease that the compound that ER is shifted to well-oxygenated environment and biopreparate may stress be relevant with ER dysfunction for treatment ER is effective.In order to investigate this possibility, the medicine of two kinds of FDA-approvals, Pioglitazone (Actos) and cow-bezoar ursodesoxycholic acid (TUDCA), can affect ER redox state and improve the cell death in ER disease cell model.Pioglitazone approval is used for the treatment of to be suffered from the patient of diabetes B and in the syndromic mouse model of Wolfram, has shown protection pancreas Instreptozotocin Induced.Pioglitazone is presented in the pancreas beta cell of processing with thapsigargin ER is shifted to (Figure 38, right figure) and protects these cells to avoid cell death (Figure 38, left figure) towards well-oxygenated environment.Another kind of little molecule, TUDCA, has been used for the treatment of gall stone and biliary cirrhosis, and shows that the ER alleviating in diabetic mice model stress.TUDCA demonstration is also avoided dead (Figure 38, left figure) by ER towards well-oxygenated environment transfer (Figure 38, right figure) Cell protection under ER stressed condition.
The reagent that ER is shifted towards well-oxygenated environment can resist ER stress this discovery of protection, allow the new shaker test of exploitation to use high flux means to differentiate the micromolecular inhibitor of new reduction ER.Carry out the tentative screening (pilot screen) of 1280-library of compounds (MicroSource), the collection (Figure 39) of 1,040 united states drug and 240 International Pharmaceuticals.In this experiment, the INS-1832/13 cell inoculation (20,000 cells/well) of stably express MEROS-GFP is in black light (black optical) 96-orifice plate.After 24 hours, use TeMo liquid handling robot to add 2 each compounds of μ L.After other 24 hours, cell stimulates 2 hours with 0.2mMDTT strong reductant, then calculates MEROS-GFP ratio.The average specific of untreated cell is 0.037 (S.D.=0.003), and the cell that DTT-processes is 0.069 (S.D.=0.006).Positive compound for can keep MEROS-GFP than lower than 0.05 those.Use this standard, in screening, identify 20 kinds of positive compounds.
Using 0.4mM palmitate (ester) and the combination of 20mM glucose to carry out second screening stress with induction ER.In twice screening, identify 9 kinds of common positive compounds, wherein 5 kinds because autofluorescence is removed.In order further to eliminate false positive, remaining 4 kinds of common compound pre-service for INS-1 832/13 cell of stably express MEROS-GFP, stimulate 24 hours with 0.5mM palmitate (ester), and use FACS to measure MEROS-GFP ratio.This other step is removed as false-positive two kinds of compounds.Reagent apomorphine and griseofulvin that remaining two kinds of compounds are clinical use.Although differentiate positively in shaker test, griseofulvin has strong toxic action to INS-1 832/13 cell.
Embodiment 6. apomorphines by ER towards well-oxygenated environment shift and to ER stress protection
Carry out other experiment to confirm that apomorphine can shift from reducing environment ER to well-oxygenated environment.In these experiments, express INS-1 832/13 cell of MEROS-GFP and process 24 hours with apomorphine, then with palmitate (ester), stimulate 24 hours.Data show that apomorphine processing makes the cell colony with reduction ER reduce (Figure 40).Cell viability in INS-1 832/13 cell of processing with palmitate (ester) and mitochondrial membrane potential are used respectively propidium iodide (PI) and MitoProbe (Invitrogen) dyeing to measure.The colony that apomorphine makes to have lower mitochondrial membrane potential reduce (Figure 41) and suppress ER stress-cell death (Figure 42) of induction.Apomorphine also protect INS-1 832/13 cell avoid by the ER of strong ER stress-induced agent thapsigargin induction stress-cell death (Figure 43) of mediation.Jointly, these data show apomorphines by ER towards well-oxygenated environment shifts and to resist ER stress protection.
Embodiment 7. shifts ER little molecule towards well-oxygenated environment can alleviate the pathology that ER stress cell model
Above-mentioned data show apomorphine and Pioglitazone have ER is shifted towards well-oxygenated environment and resist ER stress the ability of protection.Carry out other experiment with determine whether apomorphine and Pioglitazone can protect people's pancreas islet avoid ER stress-mediation cell death.Data show that apomorphine and Pioglitazone all can protect people's pancreas islet to avoid the cell death of thapsigargin-mediation (Figure 44, left figure).
Use the Doxycycline-derivable WFS1 in INS-1 832/13-source to knock out cell, the syndromic cell model of Wolfram, carries out other experiment.This model is used for testing whether apomorphine and Pioglitazone can prevent cell death.As front, report, the WFS1 of shRNA-mediation knocks out and causes cell death, and its cracking by Caspase-3 completes people such as (, Diabetologia 48:2313-2321,2005) Riggs.In this model; apomorphine and Pioglitazone have all suppressed cracking and the cracking (PARP) of poly-(the ADP)-ribosylation enzyme (ribosylating enzyme) of Caspase-3 substrate of Caspase-3; and the beta cell that can protect WFS1-to knock out is avoided cell death (Figure 44, right figure).Consider altogether, these data show that the little molecule that ER is shifted towards well-oxygenated environment can alleviate the pathology of the cell model of ER disease.
Embodiment 8. soluble M ANF protection pancreas beta cells avoid ER stress with ER stress-apoptotic cell death of induction
Carry out other experiment with determine whether soluble M ANF can when being exposed to the reagent of induction er stress, prevent the endoplasmic reticulum of pancreas beta cell in fluctuation in redox environment.Use the transfection of expressing MEROS-GFP to have INS-1 832/13 cell (pancreas beta cell system) of slow virus carrier (lentivirus vector) to test.Cell is cultivated in 11mM or 25mM glucose, and untreated or process with 0.5 μ g/mL soluble M ANF.Cell is then used facs analysis to use excitation spectrum and the emission spectrum between 520-570nM (FITC-A light filter) between 460-495nM to analyze, and wherein allows the particular detection from the fluorescent emission of the EroGFP of the reduction in transfectional cell.Data illustrate the cell quantity that processing with soluble M ANF causes containing reduction MEROS-GFP that can detection level and reduce (Figure 45; Bottom-right graph vs. lower-left figure).With above-mentioned data consistent, these data show that pancreas beta cell utilizes the processing of soluble M ANF ER can be shifted towards well-oxygenated environment, and can be used for treatment or prevent that experimenter's Pancreatic beta cells function is disorderly.
Other embodiment
It should be understood that aforementioned description is intended to explanation but does not limit scope of the present invention when the present invention describes record in detail together with it, scope of the present invention is defined by the following claims.Other side, advantage and be modified in the scope of following claim.

Claims (26)

1. a method of diagnosing experimenter's pancreas beta cell disorder, described method comprises:
The level of the neurotrophic factor (MANF) of determining solubility midbrain astroglia source in the biological sample from described experimenter; With
Level by soluble M ANF in described biological sample is compared with the reference levels of soluble M ANF,
Wherein compare the level rising of soluble M ANF in described biological sample with described reference levels and show that described experimenter suffers from the disorder of pancreas beta cell.
2. predict that experimenter develops a method for the risk of pancreas beta cell disorder, described method comprises:
The level of the neurotrophic factor (MANF) of determining solubility midbrain astroglia source in the biological sample from described experimenter; With
Level by soluble M ANF in described biological sample is compared with the reference levels of soluble M ANF,
Wherein compare the level of soluble M ANF in described biological sample with described reference levels and raise and show that described experimenter develops the risk of pancreas beta cell disorder and increase, and compare with described reference levels that the level of soluble M ANF in described biological sample reduces or not have significant difference to show that described experimenter has the risk of development pancreas beta cell disorder normal or reduce.
3. monitor experimenter's pancreas Instreptozotocin Induced or a method for pancreas beta cell group, described method comprises:
Determine neurotrophic factor (MANF) in when point very first time solubility midbrain astroglia source level in the biological sample from described experimenter;
Determine the level of soluble M ANF in the biological sample from described experimenter when the second time point; With
When during by the second time point, the level of soluble M ANF in described biological sample put with the very first time, the level of soluble M ANF in described biological sample compared,
When while wherein putting with the very first time, the level of soluble M ANF in described biological sample compared the second time point, the level of soluble M ANF in described biological sample raises and shows that described experimenter's pancreas Instreptozotocin Induced declines or the minimizing of pancreas beta cell group, the level of soluble M ANF in described biological sample reduces or shows that without marked change described experimenter's pancreas Instreptozotocin Induced does not change or improves when comparing the second time point with the level of when point very first time soluble M ANF in described biological sample, or pancreas beta cell group does not change or increases.
4. a method of selecting to treat the experimenter of pancreas beta cell disorder, described method comprises:
The level of the neurotrophic factor (MANF) of determining solubility midbrain astroglia source in the biological sample from described experimenter; With
Level by soluble M ANF in described biological sample is compared with the reference levels of soluble M ANF,
Selection has to be compared experimenter that the level of soluble M ANF in described biological sample raise to be used for the treatment of pancreas beta cell disorderly with described reference levels.
5. according to the method described in claim 1-4 any one, the group of free type 1 diabetes, diabetes B and Wolfram syndrome composition is selected in the disorder of wherein said pancreas beta cell.
6. according to the method described in claim 1-4 any one, wherein said reference levels are the threshold level of soluble M ANF.
7. according to the method described in claim 1-4 any one, wherein said reference levels are the level of soluble M ANF in health volunteer.
8. treat or delay a method for experimenter's the disorderly morbidity of pancreas beta cell, it comprises neurotrophic factor (MANF) from the solubility midbrain astroglia source that comprises the sequence same with SEQ ID NO:2 at least 80% of effective dose to described experimenter or the apomorphine of using.
9. reduce a method for er stress in pancreas B-cell, described method comprises makes described pancreas beta cell contact with neurotrophic factor (MANF) or the apomorphine in the solubility midbrain astroglia source that comprises the sequence same with SEQ ID NO:2 at least 80% of effective dose.
10. method according to claim 9, wherein said pancreas beta cell is in described experimenter.
11. 1 kinds of methods that reduce or delay the apoptotic cell death of er stress-induction in plural pancreas beta cell colony, described method comprises that neurotrophic factor (MANF) or apomorphine that described pancreas beta cell colony is originated with the solubility midbrain astroglia that comprises the sequence same with SEQ ID NO:2 at least 80% of effective dose contact.
12. methods according to claim 11, wherein said pancreas beta cell colony is in described experimenter.
13. according to the method described in claim 1-4,8,10 and 12 any one, and wherein said experimenter is the mankind.
14. 1 kinds of methods of monitoring the treatment effect of pancreas beta cell disorder in experimenter, described method comprises:
Determine neurotrophic factor (MANF) in when point very first time solubility midbrain astroglia source level in the biological sample from described experimenter;
Determine the level of soluble M ANF in the biological sample from described experimenter when the second time point; With
When during by the second time point, the level of soluble M ANF in described biological sample put with the very first time, the level of soluble M ANF in described biological sample compared,
Wherein (i) described very first time point is for before treatment, with described the second time point be the random time point after initial in treatment, or (ii) described very first time point after treatment is initial, and described the second time point be later than treatment during or time point afterwards; With
When while putting with the very first time, the level of soluble M ANF in described biological sample compared the second time point, the level reduction of soluble M ANF in described biological sample shows that described treatment is effective in described experimenter.
15. screening is used for the treatment of or delays a method for experimenter's the disorderly candidate compound of falling ill of pancreas beta cell, described method comprises:
Pancreas beta cell is provided;
Described pancreas beta cell is contacted with test compound;
Determine the level of the neurotrophic factor (MANF) in the solubility midbrain astroglia source being produced by described pancreas beta cell under the existence of described test compound; With
The level of the solubility (MANF) being produced by described pancreas beta cell under the existence of described test compound is compared with the reference levels of soluble M ANF,
The soluble M ANF being produced by described pancreas beta cell is compared in selection level to described reference levels raises relevant compound as the candidate compound that is used for the treatment of or delays described experimenter's the disorderly morbidity of pancreas beta cell.
16. method according to claim 15, wherein said reference levels are in the level that does not have the soluble M ANF being produced by pancreas beta cell under candidate agent.
17. methods according to claim 15, wherein said reference levels are the threshold level of soluble M ANF.
18. screening is used for the treatment of or delays a method for experimenter's the disorderly candidate compound of falling ill of pancreas beta cell, described method comprises:
The mammalian cell of the reporter protein of expressing the fluorescin contain BiP burst, isotope of redox-sensitive and amino acid sequence KDEL is provided;
Described cell is contacted with test compound;
Determine the amount of the reporter protein being oxidized in described cell; With
The amount of the reporter protein being oxidized in described cell is compared with reference levels;
The reporter protein being oxidized in cell is compared in selection level to described reference levels raises relevant test compound as the candidate compound that is used for the treatment of or delays described experimenter's the disorderly morbidity of pancreas beta cell.
19. methods according to claim 18, the amount that wherein said reference levels are the reporter protein that is oxidized in mammalian cell under not there is not candidate agent.
20. methods according to claim 18, wherein said reference levels are the threshold level of the reporter protein of oxidation.
21. screening is used for the treatment of or delays a method for experimenter's the disorderly candidate compound of falling ill of pancreas beta cell, described method comprises:
The mammalian cell of the reporter protein of expressing the fluorescin contain BiP burst, isotope of redox-sensitive and amino acid sequence KDEL is provided;
Described cell is contacted with test compound;
Determine the amount of the reporter protein reducing in described cell; With
The amount of the reporter protein reducing in described cell is compared with reference levels;
The reporter protein reducing in cell is compared in selection level to described reference levels reduces relevant test compound as the candidate compound that is used for the treatment of or delays experimenter's the disorderly morbidity of pancreas beta cell.
22. methods according to claim 21, the amount that wherein said reference levels are the reporter protein that reduces in mammalian cell under not there is not candidate agent.
23. methods according to claim 21, wherein said reference levels are the threshold level of the reporter protein of oxidation.
24. 1 kinds of kits, it comprises:
The antibody of specific binding soluble M ANF or antigen-binding antibody fragment; With
Be bonded at least one antibody or the antigen-binding antibody fragment of the protein of the group of selecting free insulin, C-albumen and IAPP composition.
25. 1 kinds of pharmaceutical compositions, it comprises:
The soluble M ANF albumen that comprises the sequence same with SEQ ID NO:2 at least 80%, or apomorphine; With
At least one is used for the treatment of the additives of pancreas beta cell disorder.
26. compositions according to claim 25, wherein said at least one additives are insulin.
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